Antisense modulation of estrogen receptor beta expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of estrogen receptor beta. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding estrogen receptor beta. Methods of using these compounds for modulation of estrogen receptor beta expression and for treatment of diseases associated with expression of estrogen receptor beta are provided.

INTRODUCTION

[0001] This application is a continuation of U.S. patent applicationSer. No. 10/005,058 filed Dec. 7, 2001.

FIELD OF THE INVENTION

[0002] The present invention provides compositions and methods formodulating the expression of estrogen receptor beta. In particular, thisinvention relates to compounds, particularly oligonucleotides,specifically hybridizable with nucleic acids encoding estrogen receptorbeta. Such compounds have been shown to modulate the expression ofestrogen receptor beta.

BACKGROUND OF THE INVENTION

[0003] Steroid, thyroid and retinoid hormones produce a diverse array ofphysiologic effects through the regulation of gene expression. Uponentering the cell, these hormones bind to a unique group ofintracellular nuclear receptors which have been characterized asligand-dependent transcription factors. This complex then moves into thenucleus where the receptor and its cognate ligand interact with thetranscription preinitiation complex affecting its stability andultimately, the rate of transcription of the target genes. Members ofthe nuclear receptor family share several structural features includinga central, highly conserved DNA-binding domain which targets thereceptor to specific DNA sequences known as hormone response elements(Kliewer et al., Science, 1999, 284, 757-760).

[0004] Estrogens are steroid hormones that exert a wide range of effectsthroughout the body. They are required for normal female sexualmaturation and promote growth and differentiation of the breast, uterus,fallopian tubes, vagina and ovaries. Male reproductive tissues such astestis and prostate are also estrogen target tissues. Furthermore,estrogens are important for bone maintenance and have a protective rolein the cardiovascular system. In the brain, estrogens appear to modulatethe regulation and reproduction of autonomic and reproductiveneuroendocrine systems, mood and cognition (Osterlund and Hurd, Prog.Neurobiol., 2001, 64, 251-267).

[0005] Similar to the other steroid hormone receptors, the estrogenreceptors consist of several structural domains that exhibit differentfunctional features. Both the estrogen receptor alpha and estrogenreceptor beta consist of five different domains, the N-terminal domain(called A/B), the DNA-binding domain (C), a short hinge region (E) and ashort C-terminal domain (F) (Osterlund and Hurd, Prog. Neurobiol., 2001,64, 251-267).

[0006] Estrogen receptor beta (also known as ESR2, estrogen receptor 2,ESRB and ER beta) was cloned in 1996. The DNA-binding and ligand bindingdomains of estrogen receptor beta are respectively 96% and 58% conservedrelative to estrogen receptor alpha (Mosselman et al., FEBS Lett., 1996,392, 49-53).

[0007] In 1997, Enmark et al. reported the genomic structure andexpression pattern of human estrogen receptor beta. The gene comprises 8exons spanning approximately 40 kb. It is expressed in multiple tissues,including developing spermatids of the testis, a finding of potentialrelevance for assessment of the effects of environmental estrogens onsperm counts (Enmark et al., J. Clin. Endocrinol. Metab., 1997, 82,4258-4265). Estrogen receptor beta was also found in ovarian granulosacells, indicating that estrogens also participate in the regulation offollicular growth (Enmark et al., J. Clin. Endocrinol. Metab., 1997, 82,4258-4265). The gene was mapped to chromosome 14q22-q24, a regionassociated with early onset of Alzheimer's disease, uterine leiomyomataand neoplasms of the kidney (Enmark et al., J. Clin. Endocrinol. Metab.,1997, 82, 4258-4265; Pedeutour et al., Genes Chromosomes Cancer, 1998,23, 361-366).

[0008] Since the human estrogen receptor alpha gene has been mapped tothe long arm of chromosome 6, the possibility of differential splicingof the estrogen receptor alpha gene to produce the estrogen beta isoformhas been ruled out (Enmark et al., J. Clin. Endocrinol. Metab., 1997,82, 4258-4265).

[0009] Nucleic acid sequences encoding human estrogen receptor beta andvariants of estrogen receptor beta are disclosed and claimed in U.S.Pat. No. 5,958,710, European patent applications EP0935000 and EP0798378as well as PCT publications WO 01/62793 and WO 97/09348 (Kalush et al.,2001; Kuiper et al., 1999; Kuiper et al., 1997). Additionally claimed inPCT publication WO 01/62793 are nucleic acids encoding the complement ofnucleic acids which encode variants of estrogen receptor beta and anisolated antibody that binds to the peptide of said estrogen receptorvariants (Kalush et al., 2001).

[0010] Several variants of estrogen receptor beta have been reported.Ogawa et al. have reported the cloning and characterization of a variantwhich is truncated at the C-terminal domain but has an extra 26 aminoacids due to alternative splicing (Ogawa et al., Nucleic Acids Res.,1998, 26, 3505-3512). This variant was named ER-beta-cx and identifiedas a potential inhibitor of estrogen action since it has noligand-binding activity (ogawa et al., Nucleic Acids Res., 1998, 26,3505-3512).

[0011] Five additional splice variants of estrogen receptor beta havebeen cloned and characterized by Moore et al. (Moore et al., Biochem.Biophys. Res. Commun., 1998, 247, 75-78). The mRNAs of these variants(designated ER-beta-1 through ER-beta-5) were found to displaydifferential patterns of expression in human tissues and tumor celllines (Moore et al., Biochem. Biophys. Res. Commun., 1998, 247, 75-78).

[0012] Shupnik et al. cloned the exon 2-deletion splice variant ofestrogen receptor beta (designated ER-beta-delta-2) from a humanpituitary tumor tissue and proposed that differential expression ofestrogen receptor isoforms may influence the biological properties ofthe tumors and affect their ability to respond to estrogen andantiestrogen therapies (Shupnik et al., J. Clin. Endocrinol. Metab.,1998, 83, 3965-3972).

[0013] Four additional splice variants (unpublished) have beenidentified and are herein designated ER-beta-5/6, ER-beta-7, ER-beta-8and ER-beta-9.

[0014] Ogawa et al. have shown that genetic variations at the estrogenreceptor beta locus (ESR2) may be associated with some determinants ofblood pressure and that there is a possible involvement of thispolymorphism in causing hypertension in Japanese women (Ogawa et al., J.Hum. Genet., 2000, 45, 327-330).

[0015] An antibody specific for estrogen receptor beta has been used toimmunolocalize estrogen receptor beta in histologically normal prostate,prostatic intraepithelial neoplasia, primary carcinomas and inmetastases to lymph nodes and bone. Results indicate that the presenceof estrogen receptor beta in metastatic cells may have importantimplications for the treatment of late stage disease (Leav et al., Am.J. Pathol., 2001, 159, 79-92).

[0016] A quantitative analysis of estrogen receptor alpha and beta mRNAexpression in human pancreatic adenoma tissues has indicated aprobability that estrogen receptor beta plays a more important role thanestrogen receptor alpha in pancreatic cancers (Iwao et al., Cancer Lett.(Shannon, Irel.), 2001, 170, 91-97).

[0017] Investigations of estrogen receptor beta knockout mice haveindicated that estrogen receptor beta is essential for normal ovulationbut not essential for female or male sexual differentiation, fertilityor lactation (Krege et al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95,15677-15682).

[0018] The involvement of estrogen receptor beta in cell proliferationindicates that its selective inhibition may prove to be a useful targetfor therapeutic intervention in a variety of cancers.

[0019] Because estrogens are thought to support breast cancer, estrogenantagonists have been developed which act through the estrogen receptor.Selective estrogen receptor modulators (SERMs) are small molecules whichmanifest variable agonist and antagonist properties when examined in thecontext of estrogen-dependent responses occurring in various tissues.Examples of SERMs include raloxifene and tamoxifen (Dutertre and Smith,J. Pharmacol. Exp. Ther., 2000, 295, 431-437).

[0020] Lau et al. have reported the use of an 18-mer, antisenseoligonucleotide targeting the translation start site of human estrogenreceptor beta in both normal and malignant human prostatic epithelialcells in an assessment of prostatic cells as targets forreceptor-mediated estrogenic and antiestrogenic action (Lau et al.,Cancer Res., 2000, 60, 3175-3182).

[0021] Currently, there are no known therapeutic agents that effectivelyinhibit the synthesis of estrogen receptor beta. To date, investigativestrategies aimed at modulating estrogen receptor beta expression haveinvolved the use of small molecule SERMs, an antisense oligonucleotideand gene knock-outs in mice. Consequently, there remains a long feltneed for additional agents capable of effectively inhibiting estrogenreceptor beta function.

[0022] Antisense technology is emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of expression of estrogenreceptor beta.

[0023] The present invention provides compositions and methods formodulating expression of estrogen receptor beta, including modulation ofspliced variants of estrogen receptor beta.

SUMMARY OF THE INVENTION

[0024] The present invention is directed to compounds, particularlyantisense oligonucleotides, which are targeted to a nucleic acidencoding estrogen receptor beta, and which modulate the expression ofestrogen receptor beta. Pharmaceutical and other compositions comprisingthe compounds of the invention are also provided. Further provided aremethods of modulating the expression of estrogen receptor beta in cellsor tissues comprising contacting said cells or tissues with one or moreof the antisense compounds or compositions of the invention. Furtherprovided are methods of treating an animal, particularly a human,suspected of having or being prone to a disease or condition associatedwith expression of estrogen receptor beta by administering atherapeutically or prophylactically effective amount of one or more ofthe antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding estrogen receptor beta, ultimatelymodulating the amount of estrogen receptor beta produced. This isaccomplished by providing antisense compounds which specificallyhybridize with one or more nucleic acids encoding estrogen receptorbeta. As used herein, the terms “target nucleic acid” and “nucleic acidencoding estrogen receptor beta” encompass DNA encoding estrogenreceptor beta, RNA (including pre-mRNA and mRNA) transcribed from suchDNA, and also cDNA derived from such RNA. The specific hybridization ofan oligomeric compound with its target nucleic acid interferes with thenormal function of the nucleic acid. This modulation of function of atarget nucleic acid by compounds which specifically hybridize to it isgenerally referred to as “antisense”. The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, and catalytic activity which may be engaged in orfacilitated by the RNA. The overall effect of such interference withtarget nucleic acid function is modulation of the expression of estrogenreceptor beta. In the context of the present invention, “modulation”means either an increase (stimulation) or a decrease (inhibition) in theexpression of a gene. In the context of the present invention,inhibition is the preferred form of modulation of gene expression andmRNA is a preferred target.

[0026] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding estrogen receptor beta. The targeting process alsoincludes determination of a site or sites within this gene for theantisense interaction to occur such that the desired effect, e.g.,detection or modulation of expression of the protein, will result.Within the context of the present invention, a preferred intragenic siteis the region encompassing the translation initiation or terminationcodon of the open reading frame (ORF) of the gene. Since, as is known inthe art, the translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),the translation initiation codon is also referred to as the “AUG codon,”the “start codon” or the “AUG start codon”. A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Thus, the terms “translation initiation codon” and “start codon”can encompass many codon sequences, even though the initiator amino acidin each instance is typically methionine (in eukaryotes) orformylmethionine (in prokaryotes). It is also known in the art thateukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA molecule transcribedfrom a gene encoding estrogen receptor beta, regardless of thesequence(s) of such codons.

[0027] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0028] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0029] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. It has also beenfound that introns can also be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0030] It is also known in the art that alternative RNA transcripts canbe produced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic andextronic regions.

[0031] Upon excision of one or more exon or intron regions or portionsthereof during splicing, pre-mRNA variants produce smaller “mRNAvariants”. Consequently, mRNA variants are processed pre-mRNA variantsand each unique pre-mRNA variant must always produce a unique mRNAvariant as a result of splicing. These mRNA variants are also known as“alternative splice variants”. If no splicing of the pre-mRNA variantoccurs then the pre-mRNA variant is identical to the mRNA variant.

[0032] It is also known in the art that variants can be produced throughthe use of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites.

[0033] Once one or more target sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0034] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

[0035] Antisense and other compounds of the invention which hybridize tothe target and inhibit expression of the target are identified throughexperimentation, and the sequences of these compounds are hereinbelowidentified as preferred embodiments of the invention. The target sitesto which these preferred sequences are complementary are hereinbelowreferred to as “active sites” and are therefore preferred sites fortargeting. Therefore another embodiment of the invention encompassescompounds which hybridize to these active sites.

[0036] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

[0037] For use in kits and diagnostics, the antisense compounds of thepresent invention, either alone or in combination with other antisensecompounds or therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

[0038] Expression patterns within cells or tissues treated with one ormore antisense compounds are compared to control cells or tissues nottreated with antisense compounds and the patterns produced are analyzedfor differential levels of gene expression as they pertain, for example,to disease association, signaling pathway, cellular localization,expression level, size, structure or function of the genes examined.These analyses can be performed on stimulated or unstimulated cells andin the presence or absence of other compounds which affect expressionpatterns.

[0039] Examples of methods of gene expression analysis known in the artinclude DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000,480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (reviewed in (To, Comb. Chem. High Throughput Screen, 2000, 3,235-41).

[0040] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisenseoligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. Antisenseoligonucleotide drugs, including ribozymes, have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

[0041] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0042] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about50 nucleobases (i.e. from about 8 to about 50 linked nucleosides).Particularly preferred antisense compounds are antisenseoligonucleotides, even more preferably those comprising from about 12 toabout 30 nucleobases. Antisense compounds include ribozymes, externalguide sequence (EGS) oligonucleotides (oligozymes), and other shortcatalytic RNAs or catalytic oligonucleotides which hybridize to thetarget nucleic acid and modulate its expression.

[0043] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

[0044] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0045] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0046] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;5,672,697 and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

[0047] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0048] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

[0049] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0050] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0051] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O—(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

[0052] A further prefered modification includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring thereby forming a bicyclic sugar moiety. Thelinkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

[0053] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′—OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-703 F. Similar modifications may also bemade at other positions on the oligonucleotide, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

[0054] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U) . Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyl-adenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

[0055] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

[0056] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. The compounds of the inventioncan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugates groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992 theentire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., dihexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

[0057] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0058] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

[0059] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0060] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0061] The antisense compounds of the invention are synthesized in vitroand do not include antisense compositions of biological origin, orgenetic vector constructs designed to direct the in vivo synthesis ofantisense molecules.

[0062] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes, receptortargeted molecules, oral, rectal, topical or other formulations, forassisting in uptake, distribution and/or absorption. RepresentativeUnited States patents that teach the preparation of such uptake,distribution and/or absorption assisting formulations include, but arenot limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0063] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0064] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0065] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0066] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

[0067] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0068] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of estrogen receptor betais treated by administeringantisense compounds in accordance with this invention. The compounds ofthe invention can be utilized in pharmaceutical compositions by addingan effective amount of an antisense compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the antisensecompounds and methods of the invention may also be usefulprophylactically, e.g., to prevent or delay infection, inflammation ortumor formation, for example.

[0069] The antisense compounds of the invention are useful for researchand diagnostics, because these compounds hybridize to nucleic acidsencoding estrogen receptor beta, enabling sandwich and other assays toeasily be constructed to exploit this fact. Hybridization of theantisense oligonucleotides of the invention with a nucleic acid encodingestrogen receptor betacan be detected by means known in the art. Suchmeans may include conjugation of an enzyme to the oligonucleotide,radiolabelling of the oligonucleotide or any other suitable detectionmeans. Kits using such detection means for detecting the level ofestrogen receptor beta in a sample may also be prepared.

[0070] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0071] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful. Preferred topical formulationsinclude those in which the oligonucleotides of the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Preferred lipids and liposomes include neutral (e.g.dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Oligonucleotides of the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters include but are not limited arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999 which is incorporated herein byreference in its entirety.

[0072] Compositions and formulations for oral administration includepowders or granules, microparticulates, nanoparticulates, suspensions orsolutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Preferred oralformulations are those in which oligonucleotides of the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Preferred surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Prefered bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate,. Preferedfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also prefered are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly prefered combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. application Ser. Nos. 08/886,829 (filed Jul. 1, 1997), 09/108,673(filed Jul. 1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624(filed May 21, 1998) and 09/315,298 (filed May 20, 1999) each of whichis incorporated herein by reference in their entirety.

[0073] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0074] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0075] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0076] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0077] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

[0078] Emulsions

[0079] The compositions of the present invention may be prepared andformulated as emulsions. Emulsions are typically heterogenous systems ofone liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter. (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising of two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be either water-in-oil (w/o) or of theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

[0080] Emulsions are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0081] Synthetic surfactants, also known as surface active agents, havefound wide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

[0082] Naturally occurring emulsifiers used in emulsion formulationsinclude lanolin, beeswax, phosphatides, lecithin and acacia. Absorptionbases possess hydrophilic properties such that they can soak up water toform w/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

[0083] A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0084] Hydrophilic colloids or hydrocolloids include naturally occurringgums and synthetic polymers such as polysaccharides (for example,acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, andtragacanth), cellulose derivatives (for example, carboxymethylcelluloseand carboxypropylcellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

[0085] Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

[0086] The application of emulsion formulations via dermatological, oraland parenteral routes and methods for their manufacture have beenreviewed in the literature (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199). Emulsion formulations for oral deliveryhave been very widely used because of reasons of ease of formulation,efficacy from an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

[0087] In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0088] The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

[0089] Surfactants used in the preparation of microemulsions include,but are not limited to, ionic surfactants, non-ionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

[0090] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

[0091] Microemulsions of the present invention may also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

[0092] Liposomes

[0093] There are many organized surfactant structures besidesmicroemulsions that have been studied and used for the formulation ofdrugs. These include monolayers, micelles, bilayers and vesicles.Vesicles, such as liposomes, have attracted great interest because oftheir specificity and the duration of action they offer from thestandpoint of drug delivery. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers.

[0094] Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

[0095] In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

[0096] Further advantages of liposomes include; liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

[0097] Liposomes are useful for the transfer and delivery of activeingredients to the site of action. Because the liposomal membrane isstructurally similar to biological membranes, when liposomes are appliedto a tissue, the liposomes start to merge with the cellular membranes.As the merging of the liposome and cell progresses, the liposomalcontents are emptied into the cell where the active agent may act.

[0098] Liposomal formulations have been the focus of extensiveinvestigation as the mode of delivery for many drugs. There is growingevidence that for topical administration, liposomes present severaladvantages over other formulations. Such advantages include reducedside-effects related to high systemic absorption of the administereddrug, increased accumulation of the administered drug at the desiredtarget, and the ability to administer a wide variety of drugs, bothhydrophilic and hydrophobic, into the skin.

[0099] Several reports have detailed the ability of liposomes to deliveragents including high-molecular weight DNA into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin. The majority of applicationsresulted in the targeting of the upper epidermis.

[0100] Liposomes fall into two broad classes. Cationic liposomes arepositively charged liposomes which interact with the negatively chargedDNA molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

[0101] Liposomes which are pH-sensitive or negatively-charged, entrapDNA rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

[0102] One major type of liposomal composition includes phospholipidsother than naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0103] Several studies have assessed the topical delivery of liposomaldrug formulations to the skin. Application of liposomes containinginterferon to guinea pig skin resulted in a reduction of skin herpessores while delivery of interferon via other means (e.g. as a solutionor as an emulsion) were ineffective (Weiner et al., Journal of DrugTargeting, 1992, 2, 405-410). Further, an additional study tested theefficacy of interferon administered as part of a liposomal formulationto the administration of interferon using an aqueous system, andconcluded that the liposomal formulation was superior to aqueousadministration (du Plessis et al., Antiviral Research, 1992, 18,259-265).

[0104] Non-ionic liposomal systems have also been examined to determinetheir utility in the delivery of drugs to the skin, in particularsystems comprising non-ionic surfactant and cholesterol. Non-ionicliposomal formulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

[0105] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,53, 3765). Various liposomes comprising one or more glycolipids areknown in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987,507, 64) reported the ability of monosialoganglioside G_(M1),galactocerebroside sulfate and phosphatidylinositol to improve bloodhalf-lives of liposomes. These findings were expounded upon by Gabizonet al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No.4,837,028 and WO 88/04924, both to Allen et al., disclose liposomescomprising (1) sphingomyelin and (2) the ganglioside G_(M1) or agalactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.)discloses liposomes comprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0106] Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

[0107] A limited number of liposomes comprising nucleic acids are knownin the art. WO 96/40062 to Thierry et al. discloses methods forencapsulating high molecular weight nucleic acids in liposomes. U.S.Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomesand asserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

[0108] Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

[0109] Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes. The most common wayof classifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

[0110] If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

[0111] If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

[0112] If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

[0113] If the surfactant molecule has the ability to carry either apositive or negative charge, the surfactant is classified as amphoteric.Amphoteric surfactants include acrylic acid derivatives, substitutedalkylamides, N-alkylbetaines and phosphatides.

[0114] The use of surfactants in drug products, formulations and inemulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms,Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0115] Penetration Enhancers

[0116] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

[0117] Penetration enhancers may be classified as belonging to one offive broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Eachof the above mentioned classes of penetration enhancers are describedbelow in greater detail.

[0118] Surfactants: In connection with the present invention,surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of oligonucleotidesthrough the mucosa is enhanced. In addition to bile salts and fattyacids, these penetration enhancers include, for example, sodium laurylsulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetylether) (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p.92); and perfluorochemical emulsions, such as FC-43.Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0119] Fatty acids: Various fatty acids and their derivatives which actas penetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654)

[0120] Bile salts: The physiological role of bile includes thefacilitation of dispersion and absorption of lipids and fat-solublevitamins (Brunton, Chapter 38 in: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, NewYork, 1996, pp. 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus the term “bilesalts” includes any of the naturally occurring components of bile aswell as any of their synthetic derivatives. The bile salts of theinvention include, for example, cholic acid (or its pharmaceuticallyacceptable sodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0121] Chelating Agents: Chelating agents, as used in connection withthe present invention, can be defined as compounds that remove metallicions from solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0122] Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacycloalkanone derivatives (Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, page 92); andnon-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

[0123] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

[0124] Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

[0125] Carriers

[0126] Certain compositions of the present invention also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0127] Excipients

[0128] In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

[0129] Pharmaceutically acceptable organic or inorganic excipientsuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

[0130] Formulations for topical administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

[0131] Suitable pharmaceutically acceptable excipients include, but arenot limited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

[0132] Other Components

[0133] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

[0134] Aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

[0135] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

[0136] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

[0137] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 9per kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0138] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2′-alkoxy Amidites

[0139] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylphosphoramidites were purchased from commercial sources (e.g. Chemgenes,Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxysubstituted nucleoside amidites are prepared as described in U.S. Pat.No. 5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, the standard cycle for unmodifiedoligonucleotides was utilized, except the wait step after pulse deliveryof tetrazole and base was increased to 360 seconds.

[0140] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

2′-Fluoro Amidites 2′-Fluorodeoxyadenosine Amidites

[0141] 2′-fluoro oligonucleotides were synthesized as describedpreviously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] andU.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, theprotected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine wassynthesized utilizing commercially available9-beta-D-arabinofuranosyladenine as starting material and by modifyingliterature procedures whereby the 2′-alpha-fluoro atom is introduced bya SN2-displacement of a 2′-beta-trityl group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyl groups was accomplished usingstandard methodologies and standard methods were used to obtain the5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

[0142] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

2′-Fluorouridine

[0143] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

2′-Fluorodeoxycytidine

[0144] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

2′-O-(2-Methoxyethyl) Modified Amidites

[0145] 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

[0146] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to reflux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60° C. at 1mm Hg for 24 h) to give a solid that was crushed to a light tan powder(57 g, 85% crude yield). The NMR spectrum was consistent with thestructure, contaminated with phenol as its sodium salt (ca. 5%). Thematerial was used as is for further reactions (or it can be purifiedfurther by column chromatography using a gradient of methanol in ethylacetate (10-25%) to give a white solid, mp 222-4° C.).

2′-O-Methoxyethyl-5-methyluridine

[0147] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160° C. After heating for 48 hours at 155-160°C., the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product. Additional material was obtained byreworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0148] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) wasco-evaporated with pyridine (250 mL) and the dried residue dissolved inpyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g,0.278 M) was added and the mixture stirred at room temperature for onehour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the reaction stirred for an additional one hour. Methanol (170mL) was then added to stop the reaction. HPLC showed the presence ofapproximately 70% product. The solvent was evaporated and trituratedwith CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) andextracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturatedNaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated.275 g of residue was obtained. The residue was purified on a 3.5 kgsilica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1)containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 gof product. Approximately 20 g additional was obtained from the impurefractions to give a total yield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0149] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by TLC by first quenching the TLC sample with the additionof MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0150] A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0151] A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0152] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, TLC showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ andevaporated to give a residue (96 g). The residue was chromatographed ona 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et₃NH asthe eluting solvent. The pure product fractions were evaporated to give90 g (90%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

[0153]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (TLC showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2′-O-(Aminooxyethyl) Nucleoside Amidites and2′-O-(Dimethylaminooxyethyl) Nucleoside Amidites2′-(Dimethylaminooxyethoxy) Nucleoside Amidites

[0154] 2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites] areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0155] O² -2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction.The solution was concentrated under reduced pressure to a thick oil.This was partitioned between dichloromethane (1 L) and saturated sodiumbicarbonate (2×1 L) and brine (1 L). The organic layer was dried oversodium sulfate and concentrated under reduced pressure to a thick oil.The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether(600 mL) and the solution was cooled to −10° C. The resultingcrystalline product was collected by filtration, washed with ethyl ether(3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of whitesolid. TLC and NMR were consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-0-(2-hydroxyethyl)-5-methyluridine

[0156] In a 2 L stainless steel, unstirred pressure reactor was addedborane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood andwith manual stirring, ethylene glycol (350 mL, excess) was addedcautiously at first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure<100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0157]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.639, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0158]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridineas white foam (1.95 g, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

[0159]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25mL). Ethyl acetate layer was driedover anhydrous Na₂SO₄ and evaporated to dryness. The residue obtainedwas purified by flash column chromatography and eluted with 5% MeOH inCH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0160] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH).This mixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0161] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0162] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy) Nucleoside Amidites

[0163] 2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0164] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

[0165] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known inthe art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂,or 2′-DMAEOE nucleoside amidites) are prepared as follows. Othernucleoside amidites are prepared similarly.

2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

[0166] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) isslowly added to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O²-2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine

[0167] To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine)gives the title compound.

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0168] Diisopropylaminotetrazolide (0.6 g) and2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are addedto a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2 Oligonucleotide Synthesis

[0169] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 380B) using standard phosphoramidite chemistrywith oxidation by iodine.

[0170] Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution. Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

[0171] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0172] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0173] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0174] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0175] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0176] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0177] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3 Oligonucleoside Synthesis

[0178] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0179] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0180] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4 PNA Synthesis

[0181] Peptide nucleic acids (PNAs) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5 Synthesis of Chimeric Oligonucleotides

[0182] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0183] [2′-O--Me]—[2′-deoxy]--[2′-O—Me] Chimeric PhosphorothioateOligonucleotides

[0184] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2′-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 ammonia/ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hrs at room temperatureis then done to deprotect all bases and sample was again lyophilized todryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at roomtemperature to deprotect the 2′ positions. The reaction is then quenchedwith 1M TEAA and the sample is then reduced to ½ volume by rotovacbefore being desalted on a G25 size exclusion column. The oligorecovered is then analyzed spectrophotometrically for yield and forpurity by capillary electrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[0185] [2′-O-(2-methoxyethyl)]--[2′-deoxy]--[−2′-O-(methoxyethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxyPhosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[0186] [2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxyphosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0187] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6 Oligonucleotide Isolation

[0188] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

[0189] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a standard 96 well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

[0190] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96 Well Plate Format

[0191] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

[0192] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following 5 cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,Ribonuclease protection assays, or RT-PCR.

[0193] T-24 Cells:

[0194] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum ((Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

[0195] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0196] A549 Cells:

[0197] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

[0198] NHDF Cells:

[0199] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville, Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville, Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

[0200] HEK Cells:

[0201] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville, Md.). HEKs were routinelymaintained in Keratinocyte Growth Medium (Clonetics Corporation,Walkersville, Md.) formulated as recommended by the supplier. Cells wereroutinely maintained for up to 10 passages as recommended by thesupplier.

[0202] T47D Cells:

[0203] The T47D breast adenocarcinoma cells were obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). Cells werecultured in Gibco DMEM High glucose media supplemented with 10% FBS.

[0204] Treatment with Antisense Compounds:

[0205] When cells reached 70% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.After 4-7 hours of treatment, the medium was replaced with fresh medium.Cells were harvested 16-24 hours after oligonucleotide treatment.

[0206] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration ofpositive control oligonucleotide that results in 80% inhibition ofc-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf mRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10 Analysis of Oligonucleotide Inhibition of Estrogen ReceptorBeta Expression

[0207] Antisense modulation of estrogen receptor beta expression can beassayed in a variety of ways known in the art. For example, estrogenreceptor beta mRNA levels can be quantitated by, e.g., Northern blotanalysis, competitive polymerase chain reaction (PCR), or real-time PCR(RT-PCR). Real-time quantitative PCR is presently preferred. RNAanalysis can be performed on total cellular RNA or poly(A)+ mRNA. Thepreferred method of RNA analysis of the present invention is the use oftotal cellular RNA as described in other examples herein. Methods of RNAisolation are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis isroutine in the art and is taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, JohnWiley & Sons, Inc., 1996. Real-time quantitative (PCR) can beconveniently accomplished using the commercially available ABI PRISM™7700 Sequence Detection System, available from PE-Applied Biosystems,Foster City, Calif. and used according to manufacturer's instructions.

[0208] Protein levels of estrogen receptor beta can be quantitated in avariety of ways well known in the art, such as immunoprecipitation,Western blot analysis (immunoblotting), ELISA or fluorescence-activatedcell sorting (FACS). Antibodies directed to estrogen receptor beta canbe identified and obtained from a variety of sources, such as the MSRScatalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can beprepared via conventional antibody generation methods. Methods forpreparation of polyclonal antisera are taught in, for example, Ausubel,F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp.11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation ofmonoclonal antibodies is taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5,John Wiley & Sons, Inc., 1997.

[0209] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons,Inc., 1998. Western blot (immunoblot) analysis is standard in the artand can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley& Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11 Poly(A)+ mRNA Isolation

[0210] Poly(A)+ mRNA was isolated according to Miura et al., Clin.Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolationare taught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993. Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70° C. was added to each well, the plate wasincubated on a 90° C. hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

[0211] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12 Total RNA Isolation

[0212] Total RNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia, Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 150 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 150 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a wastecollection tray and attached to a vacuum source. Vacuum was applied for1 minute. 500 μL of Buffer RWl was added to each well of the RNEASY 96™plate and incubated for 15 minutes and the vacuum was again applied for1 minute. An additional 500 μL of Buffer RWl was added to each well ofthe RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL ofBuffer RPE was then added to each well of the RNEASY 96™ plate and thevacuum applied for a period of 90 seconds. The Buffer RPE wash was thenrepeated and the vacuum was applied for an additional 3 minutes. Theplate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 170 μL water into each well, incubating1 minute, and then applying the vacuum for 3 minutes.

[0213] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

[0214] Real-Time Quantitative PCR Analysis of Estrogen Receptor BetamRNA Levels

[0215] Quantitation of estrogen receptor beta mRNA levels was determinedby real-time quantitative PCR using the ABI PRISM™ 7700 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR, in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM, obtained from either Operon Technologies Inc.,Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa)is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either Operon Technologies Inc., Alameda, Calif. orIntegrated DNA Technologies Inc., Coralville, Iowa) is attached to the3′ end of the probe. When the probe and dyes are intact, reporter dyeemission is quenched by the proximity of the 3′ quencher dye. Duringamplification, annealing of the probe to the target sequence creates asubstrate that can be cleaved by the 5′-exonuclease activity of Taqpolymerase. During the extension phase of the PCR amplification cycle,cleavage of the probe by Taq polymerase releases the reporter dye fromthe remainder of the probe (and hence from the quencher moiety) and asequence-specific fluorescent signal is generated. With each cycle,additional reporter dye molecules are cleaved from their respectiveprobes, and the fluorescence intensity is monitored at regular intervalsby laser optics built into the ABI PRISM™ 7700 Sequence DetectionSystem. In each assay, a series of parallel reactions containing serialdilutions of mRNA from untreated control samples generates a standardcurve that is used to quantitate the percent inhibition after antisenseoligonucleotide treatment of test samples.

[0216] Prior to quantitative PCR analysis, primer-probe sets specific tothe target gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

[0217] PCR reagents were obtained from Invitrogen, Carlsbad, Calif.RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCRbuffer (—MgCl2), 6.6 mM MgCl2, 375 μM each of DATP, dCTP, dCTP and dGTP,375 nM each of forward primer and reverse primer, 125 nM of probe, 4Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reversetranscriptase, and 2.5×ROX dye) to 96 well plates containing 30 μL totalRNA solution. The RT reaction was carried out by incubation for 30minutes at 48° C. Following a 10 minute incubation at 95° C. to activatethe PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carriedout: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5minutes (annealing/extension).

[0218] Gene target quantities obtained by real time RT-PCR arenormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreen™(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent from Molecular Probes. Methods of RNAquantification by RiboGreen™ are taught in Jones, L. J., et al,Analytical Biochemistry, 1998, 265, 368-374.

[0219] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipettedinto a 96-well plate containing 30 μL purified, cellular RNA. The plateis read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at480 nm and emission at 520 nm.

[0220] Probes and primers to human estrogen receptor beta were designedto hybridize to a human estrogen receptor beta sequence, using publishedsequence information (GenBank accession number AB006589.1, incorporatedherein as SEQ ID NO: 3). For human estrogen receptor beta the PCRprimers were:

[0221] forward primer: CCAACACCTGGGCACCTTT (SEQ ID NO: 4)

[0222] reverse primer: TCTTTTGAGGTTCCGCATACAG (SEQ ID NO: 5) and the PCRprobe was: FAM-TCCTTTAGTGGTCCATCGCCAGTTATCACA-TAMRA (SEQ ID NO: 6) whereFAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescentreporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) isthe quencher dye. For human GAPDH the PCR primers were:

[0223] forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7)

[0224] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and the PCRprobe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 9) where JOE(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, CA) is the quencherdye.

Example 14 Northern Blot Analysis of Estrogen Receptor Beta mRNA Levels

[0225] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0226] To detect human estrogen receptor beta, a human estrogen receptorbeta specific probe was prepared by PCR using the forward primerCCAACACCTGGGCACCTTT (SEQ ID NO: 4) and the reverse primerTCTTTTGAGGTTCCGCATACAG (SEQ ID NO: 5). To normalize for variations inloading and transfer efficiency membranes were stripped and probed forhuman glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,Palo Alto, Calif.).

[0227] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15 Antisense inhibition of human estrogen receptor betaexpression by chimeric phosphorothioate oligonucleotides having 2′-MOEwings and a deoxy gap

[0228] In accordance with the present invention, a series ofoligonucleotides were designed to target different regions of the humanestrogen receptor beta RNA, using published sequences (GenBank accessionnumber AB006589.1, incorporated herein as SEQ ID NO: 3; a concatenatedsequence of exons 1A, 2-8, 1B, 9-16, 17B, and 18 from residues38001-151000 of Genbank accession number AL161756, incorporated hereinas SEQ ID NO: 10; GenBank accession number AF047463.1, representing thevariant ER-beta-9, incorporated herein as SEQ ID NO: 11; GenBankaccession number AF061055.1, representing the variant ER-beta-5,incorporated herein as SEQ ID NO: 12; GenBank accession numberAF074598.1, representing the variant ER-beta-6, incorporated herein asSEQ ID NO: 13; GenBank accession number AF074599.1, representing thevariant ER-beta-5/6, incorporated herein as SEQ ID NO: 14; GenBankaccession number AF124790.1, representing the variant ER-beta-2,incorporated herein as SEQ ID NO: 15; residues 38001-151000 from GenBankaccession number AL161756.6, representing a partial genomic sequence ofestrogen receptor beta, incorporated herein as SEQ ID NO: 16; GenBankaccession number BE313699.1, representing the variant ER-beta-8,incorporated herein as SEQ ID NO: 17; and GenBank accession numberBF510841.1, representing the variant ER-beta-7, the complement of whichis incorporated herein as SEQ ID NO: 18). The oligonucleotides are shownin Table 1. “Target site” indicates the first (5′-most) nucleotidenumber on the particular target sequence to which the oligonucleotidebinds. All compounds in Table 1 are chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings”. The wings arecomposed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside(backbone) linkages are phosphorothioate (P═S) throughout theoligonucleotide. All cytidine residues are 5-methylcytidines. Thecompounds were analyzed for their effect on human estrogen receptor betamRNA levels by quantitative real-time PCR as described in other examplesherein. Data are averages from two experiments. If present, “N.D.”indicates “no data”. TABLE 1 Inhibition of human estrogen receptor betamRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOEwings and a deoxy gap TARGET SEQ ID TARGET SEQ ID ISIS # REGION NO SITESEQUENCE % INHIB NO 192343 5′UTR 3 497 tgtctccctcttacaaacag 16 19 1923445′UTR 11 91 cataggaggaaggtatgaaa 58 20 192345 Start 12 1gtgaccagagggtacatact 46 21 Codon 192346 Exon: 13 208ggtacatacctgtccagaac 19 22 Exon Junction 192347 Exon: 14 770ggtacataccgggaatcttc 28 23 Exon Junction 192348 Coding 15 993ccctcatcccgggaatcttc 31 24 192349 Exon: 17 231 ggactccatcttgatctctt 2425 Exon Junction 192350 Exon: 18 217 atggaccactgtcttgtaag 24 26 ExonJunction 192351 Exon 18 312 agtgtctctctgtttacagg 61 27 192352 Exon: 18350 tttctgccctcgcatgcctg 49 28 Exon Junction 192353 5′UTR 10 53gcttcccaggcaatcgccca 33 29 192354 5′UTR 10 70 tgccgccgccctgtcaggct 28 30192355 Exon: 10 189 cttggatgtcttcagaacca 16 31 Exon Junction 192356 Exon2 10 314 cttctcatctccttgaattc 8 32 192357 Exon 3 10 547agtaaatatttgtttcccac 0 33 192358 Exon 3 10 578 tgctggctactgagtttagt 3 34192359 Exon: 10 601 aggactccatcttgatctct 17 35 Exon Junction 192360 Exon4 10 717 gcacctgtaatcccagctac 70 36 192361 Exon 5 10 995cagaatttctaaagacttta 2 37 192362 Exon 5 10 1009 ctagagagttggttcagaat 1338 192363 Exon 6 10 1198 ctgtaggctacaaactacct 18 39 192364 Exon 7 101418 atcccatgttttctccactg 21 40 192365 Exon 7 10 1425cctgcacatcccatgttttc 12 41 192366 5′UTR 10 2139 tctctcaaagtacccagtcc 542 192367 5′UTR 10 2239 tgaggcagagaagttagttt 26 43 192368 5′UTR 10 2316cccaccctaagtccaatttt 23 44 192369 5′UTR 10 2378 tccaaagatggagaagcatc 1045 192370 5′UTR 10 2457 ttggtgtttagccaaaatag 17 46 192371 5′UTR 10 2465tcagctgtttggtgtttagc 0 47 192372 5′UTR 10 2475 agtaccagcctcagctgttt 2148 192373 5′UTR 10 2572 tctactcaggtggcataagg 11 49 192374 5′UTR 10 3023ccagcagcaaacgtaacctc 3 50 192375 5′UTR 10 3490 gtgggtgtccaaaaagccag 5451 192376 5′UTR 10 3521 tccgcgcttgcaactgcctc 58 52 192377 5′UTR 10 3608cgcagctcgggtggtccctc 66 53 192378 5′UTR 10 3789 gtataatggcttgcagataa 3454 192379 Start 10 3880 ttatatccatgtcttgagat 55 55 Codon 192380 Exon 910 4015 agaatgtcatggctggatat 76 56 192381 Exon: 10 4232ctgtttacaggtaaggtgtg 50 57 Exon Junction 192382 Exon: 10 4237tctctctgtttacaggtaag 64 58 Exon Junction 192383 Exon: 10 4242cagtgtctctctgtttacag 49 59 Exon Junction 192384 Exon 10 10 4316cagaagtgagcatccctctt 73 60 192385 Exon 10 10 4386 aaaggccttacatccttcac72 61 192386 Exon 10 10 4404 ttgaatgcttcttttaaaaa 26 62 192387 Exon 1210 4752 catggaggcctcggtgaagg 49 63 192388 Exon 12 10 4757atcatcatggaggcctcggt 72 64 192389 Exon 12 10 4762 gggacatcatcatggaggcc48 65 192390 Exon: 10 4841 aggctgagctccacaaagcc 51 66 Exon Junction192391 Exon 13 10 4940 gcaaagatgagcttgccggg 62 67 192392 Exon 13 10 4945ctggagcaaagatgagcttg 53 68 192393 Exon 14 10 5006 atgtcaaagatttccagaat42 69 192394 Exon: 10 5105 gggtacatactggaattgag 49 70 Exon Junction192395 Exon: 10 5112 gaccagagggtacatactgg 51 71 Exon Junction 192396Exon 15 10 5153 tgagccagcttccggctgct 45 72 192397 Exon 16 10 5300agcagatgttccatgccctt 56 73 192398 Exon 16 10 5447 ttctgggagccctctttgct25 74 192399 3′UTR 10 5750 tcctcaggataagggccttt 52 75 192400 3′UTR 105814 gcagtgaaaggaagtgtggt 32 76 192401 3′UTR 10 5925actgctccatcgttgcttca 35 77 192402 3′UTR 10 6193 gcccctcatgggtgagacat 4778 192403 3′UTR 10 6267 tctgccaaagcacaaacctc 58 79 192404 3′UTR 10 6348gaagatactgaacacagttc 61 80 192405 3′UTR 10 6429 tgttgcccatttaagtccag 5681 192406 3′UTR 10 6482 accgcacctggattcccagc 52 82 192407 3′UTR 10 6557ctggtttcaaactcctgacc 40 83 192408 3′UTR 10 6715 cggtggcccaatctcggctc 5084 192409 Exon: 16 455 accacgcaccttcagaacca 24 85 Intron Junction 192410Intron: 16 1633 acttggatgtctaagaggca 9 86 Exon Junction 192411 Intron 416 16634 atgaagatgcttaccagcca 12 87 192412 Intron 16 45173tacatttgttttacaacact 55 88 1B 192413 Intron: 16 55277gtataatggctgtaaagaaa 31 89 Exon Junction 192414 Intron: 16 58200cagtgtctctctagggagca 62 90 Exon Junction 192415 Exon: 16 81128tttttctcacctgtccagaa 43 91 Intron Junction 192416 Intron 16 87123ggacaattaattattggaaa 35 92 13 192417 Intron 16 90445tgccctcatatcaaagattg 23 93 14 192418 Exon: 16 105283gcccaggctcctgacacact 57 94 Intron Junction 192419 Intron: 16 110432aattgcttttctccccatct 57 95 Exon Junction 192420 Exon 17B 16 110739cttttctgcccttaagtaga 33 96

[0229] As shown in Table 1, SEQ ID NOs 20, 21, 23, 24, 25, 26, 27, 28,29, 30, 36, 40, 43, 44, 48, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 88, 89, 90, 91, 92, 93, 94, 95 and 96demonstrated at least 21% inhibition of human estrogen receptor betaexpression in this assay and are therefore preferred. The target sitesto which these preferred sequences are complementary are herein referredto as “active sites” and are therefore preferred sites for targeting bycompounds of the present invention.

Example 16 Western Blot Analysis of Estrogen Receptor Beta ProteinLevels

[0230] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to estrogen receptorbeta is used, with a radiolabelled or fluorescently labeled secondaryantibody directed against the primary antibody species. Bands arevisualized using a PHOSPHORIMAGER™ (Molecular Dynamics, SunnyvaleCalif.).

Example 17

[0231] It is advantageous to selectively inhibit the expression of oneor more variants of estrogen receptor beta. Consequently, in oneembodiment of the present invention are oligonucleotides thatselectively target, hybridize to, and specifically inhibit one or more,but fewer than all of the variants of estrogen receptor beta. A summaryof the target sites of the variants is shown in Table 2 and includesER-beta, incorporated herein as SEQ ID NO; 3, ER-beta-9, incorporatedherein as SEQ ID NO: 11, ER-beta-5, incorporated herein as SEQ ID NO:12, ER-beta-6, incorporated herein as SEQ ID NO: 13, ER-beta-5/6,incorporated herein as SEQ ID NO: 14, ER-beta-2, incorporated herein asSEQ ID NO: 15, ER-beta-8, incorporated herein as SEQ ID NO: 17,ER-beta-7, incorporated herein as SEQ ID NO: 18, ER-beta-3, incorporatedherein as SEQ ID NO: 97, ER-beta-4, incorporated herein as SEQ ID NO:98, ER-beta-cx, incorporated herein as SEQ ID NO: 99, andER-beta-delta-2, incorporated herein as SEQ ID NO: 100. TABLE 2Targeting of individual oligonucleotides to specific variants ofestrogen receptor beta OLIGO SEQ TARGET VARIANT SEQ ISIS # ID NO. SITEVARIANT ID NO. 192343 19 497 ER beta-cx 99 192344 20 91 ER beta 9 11192345 21 1695 ER beta 3 97 192345 21 1137 ER beta 2 15 192345 21 1 ERbeta 5 12 192345 21 1 ER beta 4 98 192345 21 2500 ER beta-cx 99 19234521 1643 ER beta 3 192345 21 1470 ER beta-delta-2 100 192346 22 208 ERbeta 6 13 192347 23 770 ER beta 5/6 14 192348 24 993 ER beta 2 15 19234925 231 ER beta 8 17 192350 26 217 ER beta 7 18 192351 27 822 ER beta 397 192351 27 403 ER beta 2 15 192351 27 1627 ER beta-cx 99 192351 27 770ER beta 3 192351 27 312 ER beta 7 18 192351 27 180 ER beta 5/6 14 19235228 1307 ER beta 2 15 192352 28 2670 ER beta-cx 99 192352 28 350 ER beta7 18 192353 29 53 ER beta-cx 99 192354 30 70 ER beta-cx 99 192355 31 189ER beta-cx 99 192355 31 5 ER beta 7 18 192356 32 314 ER beta-cx 99192356 32 130 ER beta 7 18 192357 33 178 ER beta 8 17 192358 34 209 ERbeta 8 17 192359 35 232 ER beta 8 17 192360 36 348 ER beta 8 17 19236137 546 ER beta-cx 99 192362 38 560 ER beta-cx 99 192363 39 749 ERbeta-cx 99 192364 40 968 ER beta-cx 99 192365 41 975 ER beta-cx 99192375 51 71 ER beta 3 97 192375 51 19 ER beta 3 192375 51 19 ERbeta-delta-2 100 192376 52 102 ER beta 3 97 192376 52 50 ER beta 3192376 52 50 ER beta-delta-2 100 192377 53 189 ER beta 3 97 192377 53137 ER beta 3 192377 53 137 ER beta-delta-2 100 192378 54 370 ER beta 397 192378 54 318 ER beta 3 192378 54 318 ER beta-delta-2 100 192379 55461 ER beta 3 97 192379 55 42 ER beta 2 15 192379 55 1266 ER beta-cx 99192379 55 409 ER beta 3 192379 55 409 ER beta-delta-2 100 192380 56 596ER beta 3 97 192380 56 177 ER beta 2 15 192380 56 1401 ER beta-cx 99192380 56 544 ER beta 3 192380 56 127 ER beta 9 11 192380 56 544 ERbeta-delta-2 100 192381 57 813 ER beta 3 97 192381 57 394 ER beta 2 15192381 57 1618 ER beta-cx 99 192381 57 761 ER beta 3 192381 57 303 ERbeta 7 18 192381 57 171 ER beta 5/6 14 192382 58 818 ER beta 3 97 19238258 399 ER beta 2 15 192382 58 1623 ER beta-cx 99 192382 58 766 ER beta 3192382 58 308 ER beta 7 18 192382 58 176 ER beta 5/6 14 192383 59 823 ERbeta 3 97 192383 59 404 ER beta 2 15 192383 59 1628 ER beta-cx 99 19238359 771 ER beta 3 192383 59 313 ER beta 7 18 192383 59 181 ER beta 5/6 14192384 60 897 ER beta 3 97 192384 60 478 ER beta 2 15 192384 60 1702 ERbeta-cx 99 192384 60 845 ER beta 3 192384 60 255 ER beta 5/6 14 19238561 967 ER beta 3 97 192385 61 548 ER beta 2 15 192385 61 1772 ER beta-cx99 192385 61 915 ER beta 3 192385 61 325 ER beta 5/6 14 192386 62 985 ERbeta 3 97 192386 62 566 ER beta 2 15 192386 62 1790 ER beta-cx 99 19238662 933 ER beta 3 192386 62 343 ER beta 5/6 14 192387 63 1333 ER beta 397 192387 63 914 ER beta 2 15 192387 63 2138 ER beta-cx 99 192387 631281 ER beta 3 192387 63 691 ER beta 5/6 14 192387 63 1108 ERbeta-delta-2 100 192388 64 1338 ER beta 3 97 192388 64 919 ER beta 2 15192388 64 2143 ER beta-cx 99 192388 64 1286 ER beta 3 192388 64 696 ERbeta 5/6 14 192388 64 1113 ER beta-delta-2 100 192389 65 1343 ER beta 397 192389 65 924 ER beta 2 15 192389 65 2148 ER beta-cx 99 192389 651291 ER beta 3 192389 65 701 ER beta 5/6 14 192389 65 1118 ERbeta-delta-2 100 192390 66 1422 ER beta 3 97 192390 66 79 ER beta 6 13192390 66 2227 ER beta-cx 99 192390 66 1370 ER beta 3 192390 66 1197 ERbeta-delta-2 100 192391 67 1521 ER beta 3 97 192391 67 178 ER beta 6 13192391 67 2326 ER beta-cx 99 192391 67 1469 ER beta 3 192391 67 1296 ERbeta-delta-2 100 192392 68 1526 ER beta 3 97 192392 68 183 ER beta 6 13192392 68 2331 ER beta-cx 99 192392 68 1474 ER beta 3 192392 68 1301 ERbeta-delta-2 100 192393 69 1587 ER beta 3 97 192393 69 1029 ER beta 2 15192393 69 2392 ER beta-cx 99 192393 69 1535 ER beta 3 192393 69 1362 ERbeta-delta-2 100 192394 70 1686 ER beta 3 97 192394 70 1128 ER beta 2 15192394 70 2491 ER beta-cx 99 192394 70 1634 ER beta 3 192394 70 1461 ERbeta-delta-2 100 192395 71 1693 ER beta 3 97 192395 71 1135 ER beta 2 15192395 71 2498 ER beta-cx 99 192395 71 1641 ER beta 3 192395 71 1468 ERbeta-delta-2 100 192396 72 1734 ER beta 3 97 192396 72 1176 ER beta 2 15192396 72 257 ER beta 6 13 192396 72 40 ER beta 5 12 192396 72 40 ERbeta 4 98 192396 72 2539 ER beta-cx 99 192396 72 1682 ER beta 3 19239672 819 ER beta 5/6 14 192396 72 1509 ER beta-delta-2 100 192397 73 1829ER beta 3 192397 73 966 ER beta 5/6 14 192397 73 1656 ER beta-delta-2100 192398 74 1976 ER beta 3 192398 74 1113 ER beta 5/6 14 192398 741803 ER beta-delta-2 100 192399 75 383 ER beta 4 98 192400 76 447 ERbeta 4 98 192401 77 1379 ER beta 2 15 192401 77 324 ER beta 5 12 19240177 558 ER beta 4 98 192401 77 2742 ER beta-cx 99 192401 77 422 ER beta 718 192402 78 3010 ER beta-cx 99 192403 79 3084 ER beta-cx 99 192404 803165 ER beta-cx 99 192405 81 3246 ER beta-cx 99 192406 82 3299 ERbeta-cx 99 192407 83 3374 ER beta-cx 99 192408 84 3532 ER beta-cx 99192419 95 181 ER beta 4 98 192420 96 488 ER beta 4 98

[0232]

1 96 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcgctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2atgcattctg cccccaagga 20 3 3593 DNA Homo sapiens CDS (1276)...(2763) 3cgagagggga cgctccctcc tcgtaggcgt ccacactgga gaaggaataa gatgggcgat 60tgcctgggaa gcctgacagg gcggcggcag ctgggatgct ggagaggact ggccccttga 120gttactgagt ccgatgaatg tgcttgctct gctggaggaa ccgcgctcag gttacagtca 180tcccaatatg gttctgaaga catccaagtg gagatatggc atttaaattc atgagattgg 240atgagatccc accaaaggaa caggtttagg tggagacaac caaataccga tgcctaggac 300actgcagtgt ttagaattca aggagatgag aaggaaacag gagggaagat tgaaaagaag 360agtccagtgt gttatgagga aaaccccaag agcatgctgc cttacaagac aggtgaaaaa 420tgtgttctgt gaaagaaaga gtaattaact gttaaatgtt acagactgat caaataaaat 480gaagactgag aatggcctgt ttgtaagagg gagacatcaa cctgttgtgg aaaagaatga 540tcacttaaag tctttagaaa ttctgaacca actctctagc aggtgatcct tgttagaatt 600tgagccctta acgctatcca ggactggagg ttgaagggac gatagaggga gcaggaggag 660aatgcacatg gattaaggag cgagaacaca gaaatcctgg gctctcttct cccagccaca 720aggttaggtt gaaaaacaga gcagatggag gtagtttgta gcctacaggt gccctgaatg 780aagcttccac agtgctaaag tggaagaacg agggactcca agggaaggat tcaaggctgg 840gcccatgcac tgtgtaattc agaagagacc ccagaggaga tcagcgccct ctaattagcc 900ctgtatctgg gctctacagg acagacatgc ctccatttat gcaacaaata agaacagcat 960ctcatgacag tggagaaaac atgggatgtg caggtagggt tttgttttgc ctcttggtag 1020tttctttcct acggaaaatt ctccctctga tctttccaag tcaaaggctt cagcaaacat 1080ttgttgaacg cgtggattgt gtgctaggtg ggtgttatgg accatggaga atgctagaga 1140tgtaagacat gcgctgtcca atcgcagcgc aggttgtgtt gacagccatt atacttgccc 1200acgaatcttt gagaacatta taatgacctt tgtgcctctt cttgcaaggt gttttctcag 1260ctgttatctc aagac atg gat ata aaa aac tca cca tct agc ctt aat tct 1311Met Asp Ile Lys Asn Ser Pro Ser Ser Leu Asn Ser 1 5 10 cct tcc tcc tacaac tgc agt caa tcc atc tta ccc ctg gag cac ggc 1359 Pro Ser Ser Tyr AsnCys Ser Gln Ser Ile Leu Pro Leu Glu His Gly 15 20 25 tcc ata tac ata ccttcc tcc tat gta gac agc cac cat gaa tat cca 1407 Ser Ile Tyr Ile Pro SerSer Tyr Val Asp Ser His His Glu Tyr Pro 30 35 40 gcc atg aca ttc tat agccct gct gtg atg aat tac agc att ccc agc 1455 Ala Met Thr Phe Tyr Ser ProAla Val Met Asn Tyr Ser Ile Pro Ser 45 50 55 60 aat gtc act aac ttg gaaggt ggg cct ggt cgg cag acc aca agc cca 1503 Asn Val Thr Asn Leu Glu GlyGly Pro Gly Arg Gln Thr Thr Ser Pro 65 70 75 aat gtg ttg tgg cca aca cctggg cac ctt tct cct tta gtg gtc cat 1551 Asn Val Leu Trp Pro Thr Pro GlyHis Leu Ser Pro Leu Val Val His 80 85 90 cgc cag tta tca cat ctg tat gcggaa cct caa aag agt ccc tgg tgt 1599 Arg Gln Leu Ser His Leu Tyr Ala GluPro Gln Lys Ser Pro Trp Cys 95 100 105 gaa gca aga tcg cta gaa cac acctta cct gta aac aga gag aca ctg 1647 Glu Ala Arg Ser Leu Glu His Thr LeuPro Val Asn Arg Glu Thr Leu 110 115 120 aaa agg aag gtt agt ggg aac cgttgc gcc agc cct gtt act ggt cca 1695 Lys Arg Lys Val Ser Gly Asn Arg CysAla Ser Pro Val Thr Gly Pro 125 130 135 140 ggt tca aag agg gat gct cacttc tgc gct gtc tgc agc gat tac gca 1743 Gly Ser Lys Arg Asp Ala His PheCys Ala Val Cys Ser Asp Tyr Ala 145 150 155 tcg gga tat cac tat gga gtctgg tcg tgt gaa gga tgt aag gcc ttt 1791 Ser Gly Tyr His Tyr Gly Val TrpSer Cys Glu Gly Cys Lys Ala Phe 160 165 170 ttt aaa aga agc att caa ggacat aat gat tat att tgt cca gct aca 1839 Phe Lys Arg Ser Ile Gln Gly HisAsn Asp Tyr Ile Cys Pro Ala Thr 175 180 185 aat cag tgt aca atc gat aaaaac cgg cgc aag agc tgc cag gcc tgc 1887 Asn Gln Cys Thr Ile Asp Lys AsnArg Arg Lys Ser Cys Gln Ala Cys 190 195 200 cga ctt cgg aag tgt tac gaagtg gga atg gtg aag tgt ggc tcc cgg 1935 Arg Leu Arg Lys Cys Tyr Glu ValGly Met Val Lys Cys Gly Ser Arg 205 210 215 220 aga gag aga tgt ggg taccgc ctt gtg cgg aga cag aga agt gcc gac 1983 Arg Glu Arg Cys Gly Tyr ArgLeu Val Arg Arg Gln Arg Ser Ala Asp 225 230 235 gag cag ctg cac tgt gccggc aag gcc aag aga agt ggc ggc cac gcg 2031 Glu Gln Leu His Cys Ala GlyLys Ala Lys Arg Ser Gly Gly His Ala 240 245 250 ccc cga gtg cgg gag ctgctg ctg gac gcc ctg agc ccc gag cag cta 2079 Pro Arg Val Arg Glu Leu LeuLeu Asp Ala Leu Ser Pro Glu Gln Leu 255 260 265 gtg ctc acc ctc ctg gaggct gag ccg ccc cat gtg ctg atc agc cgc 2127 Val Leu Thr Leu Leu Glu AlaGlu Pro Pro His Val Leu Ile Ser Arg 270 275 280 ccc agt gcg ccc ttc accgag gcc tcc atg atg atg tcc ctg acc aag 2175 Pro Ser Ala Pro Phe Thr GluAla Ser Met Met Met Ser Leu Thr Lys 285 290 295 300 ttg gcc gac aag gagttg gta cac atg atc agc tgg gcc aag aag att 2223 Leu Ala Asp Lys Glu LeuVal His Met Ile Ser Trp Ala Lys Lys Ile 305 310 315 ccc ggc ttt gtg gagctc agc ctg ttc gac caa gta cgg ctc ttg gag 2271 Pro Gly Phe Val Glu LeuSer Leu Phe Asp Gln Val Arg Leu Leu Glu 320 325 330 agc tgt tgg atg gaggtg tta atg atg ggg ctg atg tgg cgc tca att 2319 Ser Cys Trp Met Glu ValLeu Met Met Gly Leu Met Trp Arg Ser Ile 335 340 345 gac cac ccc ggc aagctc atc ttt gct cca gat ctt gtt ctg gac agg 2367 Asp His Pro Gly Lys LeuIle Phe Ala Pro Asp Leu Val Leu Asp Arg 350 355 360 gat gag ggg aaa tgcgta gaa gga att ctg gaa atc ttt gac atg ctc 2415 Asp Glu Gly Lys Cys ValGlu Gly Ile Leu Glu Ile Phe Asp Met Leu 365 370 375 380 ctg gca act acttca agg ttt cga gag tta aaa ctc caa cac aaa gaa 2463 Leu Ala Thr Thr SerArg Phe Arg Glu Leu Lys Leu Gln His Lys Glu 385 390 395 tat ctc tgt gtcaag gcc atg atc ctg ctc aat tcc agt atg tac cct 2511 Tyr Leu Cys Val LysAla Met Ile Leu Leu Asn Ser Ser Met Tyr Pro 400 405 410 ctg gtc aca gcgacc cag gat gct gac agc agc cgg aag ctg gct cac 2559 Leu Val Thr Ala ThrGln Asp Ala Asp Ser Ser Arg Lys Leu Ala His 415 420 425 ttg ctg aac gccgtg acc gat gct ttg gtt tgg gtg att gcc aag agc 2607 Leu Leu Asn Ala ValThr Asp Ala Leu Val Trp Val Ile Ala Lys Ser 430 435 440 ggc atc tcc tcccag cag caa tcc atg cgc ctg gct aac ctc ctg atg 2655 Gly Ile Ser Ser GlnGln Gln Ser Met Arg Leu Ala Asn Leu Leu Met 445 450 455 460 ctc ctg tcccac gtc agg cat gcg agg gca gaa aag gcc tct caa aca 2703 Leu Leu Ser HisVal Arg His Ala Arg Ala Glu Lys Ala Ser Gln Thr 465 470 475 ctc acc tcattt gga atg aag atg gag act ctt ttg cct gaa gca acg 2751 Leu Thr Ser PheGly Met Lys Met Glu Thr Leu Leu Pro Glu Ala Thr 480 485 490 atg gag cagtga ccctctaatc aactcggtgg cctaaagaaa aatcttgggt 2803 Met Glu Gln 495aacattttca cttcaatttc cctctgggat cattgtaatc catgaaaaaa ataattttaa 2863agaaagagtt aaaatacttt gaagttagtt atgtggttaa aaaccacctt cctttctatt 2923atcaatccaa caatttgata actgtaaacg ctaaagtgaa gacggattct cttcagatgg 2983tctccttaac tgcccagggc ttgcagatgt ctcacccatg aggggcacca atgtagaaag 3043ctgaggcttc atctactgat gagcttcact ggtttcccct gaggtttgtg ctttggcaga 3103gaaggggagg aggggactgg gattgtgtgg tcagctgtgg ctgccaacag atgcaggtta 3163ggaactgtgt tcagtatctt ccaataagaa aggggaaatg ccgatgccta tcctctttgt 3223ttaggtagaa agtaaaatgc tactggactt aaatgggcaa caaggggctt tgcctgttca 3283tttgccatgg agagggctgg gaatccaggt gcggtggctc acacctgtaa tcccaacact 3343ttgggaggcc gaggtgggca gatcagttga ggtcaggagt ttgaaaccag cctggccaac 3403atggcgaaac cccgtctcta ttaaaaatat aataattagc caggcatggt ggtgtgtgct 3463tgtaatccca gctactcagg aggctgaggc atgagaatgg cttgaacctg gaaggcaaag 3523gttgcagtga gccgagattg ggccaccgca ctccagcctg ggtgactgac agagtgagac 3583ttgtcaaaaa 3593 4 19 DNA Artificial Sequence PCR Primer 4 ccaacacctgggcaccttt 19 5 22 DNA Artificial Sequence PCR Primer 5 tcttttgaggttccgcatac ag 22 6 30 DNA Artificial Sequence PCR Probe 6 tcctttagtggtccatcgcc agttatcaca 30 7 19 DNA Artificial Sequence PCR Primer 7gaaggtgaag gtcggagtc 19 8 20 DNA Artificial Sequence PCR Primer 8gaagatggtg atgggatttc 20 9 20 DNA Artificial Sequence PCR Probe 9caagcttccc gttctcagcc 20 10 6777 DNA Homo sapiens 5′UTR (1)...(3889)exonexon junction (199)...(200) exon 1Aexon 2 10 cgagagggga cgctccctcctcgtaggcgt ccacactgga gaaggaataa gatgggcgat 60 tgcctgggaa gcctgacagggcggcggcag ctgggatgct ggagaggact ggccccttga 120 gttactgagt ccgatgaatgtgcttgctct gctggaggaa ccgcgctcag gttacagtca 180 tcccaatatg gttctgaagacatccaagtg gagatatggc atttaaattc atgagattgg 240 atgagatccc accaaaggaacaggtttagg tggagacaac caaataccga tgcctaggac 300 actgcagtgt ttagaattcaaggagatgag aaggaaacag gagggaagat tgaaaagaag 360 agtccagtgt gttatgaggaaaaccccaag agcatgctgc cttacaagac aggtgaaaaa 420 tgtgttctgt gaaagaaagagtaattaact gttaaatgtt acagactgat caaataaaat 480 gaagactgag aatggcctgtttgtaagatc acttttaaaa ggaaaacata ggagcctgaa 540 acagaagtgg gaaacaaatatttactcaaa ctaagagact aaactcagta gccagcaaca 600 agagatcaag atggagtcctcctctgtcac ccaggctgga acgcagtggt atgatctcgg 660 ctaactgcaa cctcagcctgccaggttcaa gcaattcttc tgcctcagcc tcccgagtag 720 ctgggattac aggtgcctgctgccatgatg attaatttta tgtgttaact tagctgggct 780 gtgttgccca gatagttggttaaacattat tctggatgtt tctgtgaaga tgtttttgga 840 tgaggttaac atttagatcggtggactttg agtaaagcag attacctttc ataatttggg 900 tggggctcat ccaatcagttgaacatctga agagaccaaa agactgacct tctgcaaggg 960 agacatcaac ctgttgtggaaaagaatgat cacttaaagt ctttagaaat tctgaaccaa 1020 ctctctagca ggtgatccttgttagaattt gagcccttaa cgctatccag gactggaggt 1080 tgaagggacg atagagggagcaggaggaga atgcacatgg attaaggagc gagaacacag 1140 aaatcctggg ctctcttctcccagccacaa ggttaggttg aaaaacagag cagatggagg 1200 tagtttgtag cctacaggtgccctgaatga agcttccaca gtgctaaagt ggaagaacga 1260 gggactccaa gggaaggattcaaggctggg cccatgcacc tgtgtaattc agaagagacc 1320 ccagaggaga tcagcgccctctaattagcc ctgtatctgg gctctacagg acagacatgc 1380 ctccatttat gcaacaaataagaacagcat ctcatgacag tggagaaaac atgggatgtg 1440 caggtagggt tttgttttgcctcttggtag tttctttcct acggaaaatt ctccctctga 1500 tctttccaag tcaaaggcttcagcaaacat ttgttgaacg cgtggattgt gctaggtggg 1560 tgttatggac catggagaatgctagagatg taagacatgc gctgtccaat cgcagcgcag 1620 gttgtgttga cagtattgatagatgcattt tcttcaccct cacctatctt tttctgcctg 1680 ttggcttatg gttgaaattccttcatgacg gtttccattt ccagagatat cttgttaaca 1740 agtatatacc accaaatgaagctgattttt tttttttttt ttttttttga gacagagtct 1800 cgctctgtcg cccaggctggaatgcagtgg cgcgatcttg gctcactgca acctccgcct 1860 cccatgttca agcgattctcctgcctcagc ctcctgagta gctgggatta ctggcatgtg 1920 ccaccacgtc cagccaatttttgtattttt agtagagacg aggtttcacc atgttggtca 1980 ggctggtctc aaactcctgacctcgtgatc cacctgcctc ggcctcccaa agtgctgaga 2040 ttataggtgt gagccaccatgcctggccat gaagctgatt tttttaaacc atcatttaac 2100 attttctcca taaggtggcaaggaggaaga gcatatgggg actgggtact ttgagagacc 2160 ccaggacagg agacagggaggctgagattg gcatgttgtc tgctgcagtt atttgccagc 2220 gacacactct tcccgtccaaactaacttct ctgcctcaag gacagggaga ctctgccttt 2280 caacctgaga gaaaccaggactctcagctt taatgaaaat tggacttagg gtggggcagt 2340 ggagactttt cacagctattgtttagctga tgaagcagat gcttctccat ctttggagcc 2400 tgtcttcatt acctgtggacctcatcttta tcaacccaga gcacacttgc gtctctctat 2460 tttggctaaa caccaaacagctgaggctgg tactgtaaaa ctttccctcc aaatgccccc 2520 cctcgtcttc ctctattagagatctggatc acaaccctca aaaaccatgt cccttatgcc 2580 acctgagtag atggtttgatgattaattag gcacagatgt gacactgggg ggttctcaca 2640 atggcctgtg ggtcacatgctactttcctt ttcattttca tcagcaacag ctgccttaaa 2700 gccagttaag actgtggtcctagtctcgca ccctggggct cctgctgggg tgggtgaggg 2760 gaacacccca ttaagctgggggaactgggg ctgccaccag ggggcgcgag gggccttcgc 2820 ccgagaagag gggtgggcaggtgcctccag cggagaaggg cgccgtggcc ggaggcacag 2880 gtctccccgg tgccacttcaagtgagttcg aggaagtacc tgggatcttt gatctaacgc 2940 gaaaggcctt cccagtgacctcttgagagc tgagaaccca ctccctccac ctctagtcca 3000 cggctttgcc actccagggcccgaggttac gtttgctgct ggggatttga caaacccaaa 3060 gcctctctgg tttcaccactggctccttag aatcagacat ctgttctgaa tgacacttat 3120 gtgagtcagg ggctgaggacgtgatcctcg aagtgtggtc cccagactgg ctgtatcagt 3180 gtcggcatcc cccaggacctggttggaaat gcatattctc aggccctact ccagacctct 3240 taaatctgag actggggctgcggggagcgc catctgtgcg ccactatcct tgtgggtgga 3300 ccaggagtcg gttcgagggtgctcccactt agaggtcacg cgcggcgtcg ggcgttcctg 3360 agaccgtcgg gctccctggctcggtcacgt gggctcaggc actactcccc tctaccctcc 3420 tctcggtctt taaaaggaagaaggggctta tcgttaagtc gcttgtgatc ttttcagttt 3480 ctccagctgc tggctttttggacacccact cccccgccag gaggcagttg caagcgcgga 3540 ggctgcgaga aataactgcctcttgaaact tgcagggcga agagcaggcg gcgagcgctg 3600 ggccggggag ggaccacccgagctgcgacg ggctctgggg ctgcggggca gggctggcgc 3660 ccggagcctg agctgcaggaggtgcgctcg ctttcctcaa caggtggcgg cggggcgcgc 3720 gccgggagac cccccctaatgcgggaaaag cacgtgtccg cattttagag aaggcaaggc 3780 cggtgtgttt atctgcaagccattatactt gcccacgaat ctttgagaac attataatga 3840 cctttgtgcc tcttcttgcaaggtgttttc tcagctgtta tctcaagaca tggatataaa 3900 aaactcacca tctagccttaattctccttc ctcctacaac tgcagtcaat ccatcttacc 3960 cctggagcac ggctccatatacataccttc ctcctatgta gacagccacc atgaatatcc 4020 agccatgaca ttctatagccctgctgtgat gaattacagc attcccagca atgtcactaa 4080 cttggaaggt gggcctggtcggcagaccac aagcccaaat gtgttgtggc caacacctgg 4140 gcacctttct cctttagtggtccatcgcca gttatcacat ctgtatgcgg aacctcaaaa 4200 gagtccctgg tgtgaagcaagatcgctaga acacacctta cctgtaaaca gagagacact 4260 gaaaaggaag gttagtgggaaccgttgcgc cagccctgtt actggtccag gttcaaagag 4320 ggatgctcac ttctgcgctgtctgcagcga ttacgcatcg ggatatcact atggagtctg 4380 gtcgtgtgaa ggatgtaaggccttttttaa aagaagcatt caaggacata atgattatat 4440 ttgtccagct acaaatcagtgtacaatcga taaaaaccgg cgcaagagct gccaggcctg 4500 ccgacttcgg aagtgttacgaagtgggaat ggtgaagtgt ggctcccgga gagagagatg 4560 tgggtaccgc cttgtgcggagacagagaag tgccgacgag cagctgcact gtgccggcaa 4620 ggccaagaga agtggcggccacgcgccccg agtgcgggag ctgctgctgg acgccctgag 4680 ccccgagcag ctagtgctcaccctcctgga ggctgagccg ccccatgtgc tgatcagccg 4740 ccccagtgcg cccttcaccgaggcctccat gatgatgtcc ctgaccaagt tggccgacaa 4800 ggagttggta cacatgatcagctgggccaa gaagattccc ggctttgtgg agctcagcct 4860 gttcgaccaa gtgcggctcttggagagctg ttggatggag gtgttaatga tggggctgat 4920 gtggcgctca attgaccaccccggcaagct catctttgct ccagatcttg ttctggacag 4980 ggatgagggg aaatgcgtagaaggaattct ggaaatcttt gacatgctcc tggcaactac 5040 ttcaaggttt cgagagttaaaactccaaca caaagaatat ctctgtgtca aggccatgat 5100 cctgctcaat tccagtatgtaccctctggt cacagcgacc caggatgctg acagcagccg 5160 gaagctggct cacttgctgaacgccgtgac cgatgctttg gtttgggtga ttgccaagag 5220 cggcatctcc tcccagcagcaatccatgcg cctggctaac ctcctgatgc tcctgtccca 5280 cgtcaggcat gcgagtaacaagggcatgga acatctgctc aacatgaagt gcaaaaatgt 5340 ggtcccagtg tatgacctgctgctggagat gctgaatgcc cacgtgcttc gcgggtgcaa 5400 gtcctccatc acggggtccgagtgcagccc ggcagaggac agtaaaagca aagagggctc 5460 ccagaaccca cagtctcagtgacgcctggc cctgaggtga actggcccac agaggtcaca 5520 ggctgaagcg tgaactccagtgtgtcagga tggggagaaa agcaattcat tcatttgaag 5580 ttatcttagt gccaagagtcatgtgaaaat gtcccttgca tgtgggcaat gaaagatttg 5640 cagacgatat aaaacccagactacctcata aaagagtttt gggaatacac tgagctttga 5700 gtgaaagaag ctgcagtggcctccctggag atggggagca aaccagctta aaggccctta 5760 tcctgaggaa gagacaaaaattgacatgca caatattaag ctttgaaatg cagaccacac 5820 ttcctttcac tgcaactttgacttgtcccg catctctact taagggcaga aaaggcctct 5880 caaacactca cctcatttggaatgaagatg gagactcttt tgcctgaagc aacgatggag 5940 cagtgaccct ctaatcaactcggtggccta aagaaaaatc ttgggtaaca ttttcacttc 6000 agtttccctc tgggatcattgtaatccatg aaaaaaataa ttttaaagaa agagttaaaa 6060 tactttgaag ttagttatgtggttaaaaac caccttcctt tctattatca atccaacaat 6120 ttgataactg taaacgctaaagtgaagacg gattctcttc agatggtctc cttaactgcc 6180 cagggcttgc agatgtctcacccatgaggg gcaccaatgt agaaagctga ggcttcatct 6240 actgatgagc ttcactggtttcccctgagg tttgtgcttt ggcagagaag gggaggaggg 6300 gactgggatt gtgtggtcagctgtgcctgc caacagatgc aggttaggaa ctgtgttcag 6360 tatcttccaa taagaaaggggaaatgccga tgcctatcct ctttgtttag gtagaaagta 6420 aaatgctact ggacttaaatgggcaacaag gggctttgcc tgttcatttg ccatggagag 6480 ggctgggaat ccaggtgcggtggctcacac ctgtaatccc aacactttgg gaggccgagg 6540 tgggcagatc agttgaggtcaggagtttga aaccagcctg gccaacatgg cgaaaccccg 6600 tctctattaa aaatataataattagccagg catggtggtg tgtgcttgta atcccagcta 6660 ctcaggaggc tgaggcatgagaatggcttg aacctggaag gcaaaggttg cagtgagccg 6720 agattgggcc accgcactccagcctgggtg actgacagag tgagactctg tcaaaaa 6777 11 202 DNA Homo sapiensCDS (137)...(202) 11 ccttcccttc cgattgcatt tttctctttt cttttgctgggtgttctttc ttcttcatct 60 tttttctgtt ctgctttttc cttttttttt tttcataccttcctcctatg tagacagcca 120 ccatgaatat ccagcc atg aca ttc tat agc cct gctgtg atg aat tac agc 172 Met Thr Phe Tyr Ser Pro Ala Val Met Asn Tyr Ser1 5 10 att ccc agc aat gtc act aac ttg gaa ggt 202 Ile Pro Ser Asn ValThr Asn Leu Glu Gly 15 20 12 372 DNA Homo sapiens Start codon (4)...(6)12 agtatgtacc ctctggtcac agcgacccag gatgctgaca gcagccggaa gctggctcac 60ttgctgaacg ccgtgaccga tgctttggtt tgggtgattg ccaagagcgg catctcctcc 120cagcagcaat ccatgcgcct ggctaacctc ctgatgctcc tgtcccacgt caggcatgcg 180aggtacgcgc cctaaggagc tgctctgctt gggcttggga tgggattatg tgctccacgg 240agggtgaagt gatttgggaa aagtgtctgc aagttaagga aaatgaatgc ctcatttgga 300atgaagatgg agactctttt gcctgaagca acgatggagc agtgaccctc taatcaactc 360ggtggcctaa ag 372 13 306 DNA Homo sapiens exonexon junction(218)...(219) exon 13exon 15 13 gcctccatga tgatgtccct gaccaagttggccgacaagg agttggtaca catgatcagc 60 tgggccaaga agattcccgg ctttgtggagctcagcctgt tcgaccaagt gcggctcttg 120 gagagctgtt ggatggaggt gttaatgatggggctgatgt ggcgctcaat tgaccacccc 180 ggcaagctca tctttgctcc agatcttgttctggacaggt atgtaccctc tggtcacagc 240 gacccaggat gctgacagca gccggaagctggctcacttg ctgaacgccg tgaccgatgc 300 tttggt 306 14 1215 DNA Homo sapiensexonexon junction (780)...(781) exon 12exon 15 14 ttcccagcaa tgtcactaacttggaaggtg ggcctggtcg gcagaccaca agcccaaatg 60 tgttgtggcc aacacctgggcacctttctc ctttagtggt ccatcgccag ttatcacatc 120 tgtatgcgga acctcaaaagagtccctggt gtgaagcaag atcgctagaa cacaccttac 180 ctgtaaacag agagacactgaaaaggaagg ttagtgggaa ccgttgcgcc agccctgtta 240 ctggtccagg ttcaaagagggatgctcact tctgcgctgt ctgcagcgat tacgcatcgg 300 gatatcacta tggagtctggtcgtgtgaag gatgtaaggc cttttttaaa agaagcattc 360 aaggacataa tgattatatttgtccagcta caaatcagtg tacaatcgat aaaaaccggc 420 gcaagagctg ccaggcctgccgacttcgga agtgttacga agtgggaatg gtgaagtgtg 480 gctcccggag agagagatgtgggtaccgcc ttgtgcggag acagagaagt gccgacgagc 540 agctgcactg tgccggcaaggccaagagaa gtggcggcca cgcgccccga gtgcgggagc 600 tgctgctgga cgccctgagccccgagcagc tagtgctcac cctcctggag gctgagccgc 660 cccatgtgct gatcagccgccccagtgcgc ccttcaccga ggcctccatg atgatgtccc 720 tgaccaagtt ggccgacaaggagttggtac acatgatcag ctgggccaag aagattcccg 780 gtatgtaccc tctggtcacagcgacccagg atgctgacag cagccggaag ctggctcact 840 tgctgaacgc cgtgaccgatgctttggttt gggtgattgc caagagcggc atctcctccc 900 agcagcaatc catgcgcctggctaacctcc tgatgctcct gtcccacgtc aggcatgcga 960 gtaacaaggg catggaacatctgctcaaca tgaagtgcaa aaatgtggtc ccagtgtatg 1020 acctgctgct ggagatgctgaatgcccacg tgcttcgcgg gtgcaagtcc tccatcacgg 1080 ggtccgagtg cagcccggcagaggacagta aaagcaaaga gggctcccag aacccacagt 1140 ctcagtgacg cctggccctgaggtgaactg gcccacagag gtcacaagct gaagcgtgaa 1200 ctccagtgtg tcagg 121515 1427 DNA Homo sapiens CDS (52)...(1023) 15 gacctttgtg cctcttcttgcaaggtgttt tctcagctgt tatctcaaga c atg gat 57 Met Asp 1 ata aaa aac tcacca tct agc ctt aat tct cct tcc tcc tac aac tgc 105 Ile Lys Asn Ser ProSer Ser Leu Asn Ser Pro Ser Ser Tyr Asn Cys 5 10 15 agt caa tcc atc ttaccc ctg gag cac ggc tcc ata tac ata cct tcc 153 Ser Gln Ser Ile Leu ProLeu Glu His Gly Ser Ile Tyr Ile Pro Ser 20 25 30 tcc tat gta gac agc caccat gaa tat cca gcc atg aca ttc tat agc 201 Ser Tyr Val Asp Ser His HisGlu Tyr Pro Ala Met Thr Phe Tyr Ser 35 40 45 50 cct gct gtg atg aat tacagc att ccc agc aat gtc act aac ttg gaa 249 Pro Ala Val Met Asn Tyr SerIle Pro Ser Asn Val Thr Asn Leu Glu 55 60 65 ggt ggg cct ggt cgg cag accaca agc cca aat gtg ttg tgg cca aca 297 Gly Gly Pro Gly Arg Gln Thr ThrSer Pro Asn Val Leu Trp Pro Thr 70 75 80 cct ggg cac ctt tct cct tta gtggtc cat cgc cag tta tca cat ctg 345 Pro Gly His Leu Ser Pro Leu Val ValHis Arg Gln Leu Ser His Leu 85 90 95 tat gcg gaa cct caa aag agt ccc tggtgt gaa gca aga tcg cta gaa 393 Tyr Ala Glu Pro Gln Lys Ser Pro Trp CysGlu Ala Arg Ser Leu Glu 100 105 110 cac acc tta cct gta aac aga gag acactg aaa agg aag gtt agt ggg 441 His Thr Leu Pro Val Asn Arg Glu Thr LeuLys Arg Lys Val Ser Gly 115 120 125 130 aac cgt tgc gcc agc cct gtt actggt cca ggt tca aag agg gat gct 489 Asn Arg Cys Ala Ser Pro Val Thr GlyPro Gly Ser Lys Arg Asp Ala 135 140 145 cac ttc tgc gct gtc tgc agc gattac gca tcg gga tat cac tat gga 537 His Phe Cys Ala Val Cys Ser Asp TyrAla Ser Gly Tyr His Tyr Gly 150 155 160 gtc tgg tcg tgt gaa gga tgt aaggcc ttt ttt aaa aga agc att caa 585 Val Trp Ser Cys Glu Gly Cys Lys AlaPhe Phe Lys Arg Ser Ile Gln 165 170 175 gga cat aat gat tat att tgt ccagct aca aat cag tgt aca atc gat 633 Gly His Asn Asp Tyr Ile Cys Pro AlaThr Asn Gln Cys Thr Ile Asp 180 185 190 aaa aac cgg cgc aag agc tgc caggcc tgc cga ctt cgg aag tgt tac 681 Lys Asn Arg Arg Lys Ser Cys Gln AlaCys Arg Leu Arg Lys Cys Tyr 195 200 205 210 gaa gtg gga atg gtg aag tgtggc tcc cgg aga gag aga tgt ggg tac 729 Glu Val Gly Met Val Lys Cys GlySer Arg Arg Glu Arg Cys Gly Tyr 215 220 225 cgc ctt gtg cgg aga cag agaagt gcc gac gag cag ctg cac tgt gcc 777 Arg Leu Val Arg Arg Gln Arg SerAla Asp Glu Gln Leu His Cys Ala 230 235 240 ggc aag gcc aag aga agt ggcggc cac gcg ccc cga gtg cgg gag ctg 825 Gly Lys Ala Lys Arg Ser Gly GlyHis Ala Pro Arg Val Arg Glu Leu 245 250 255 ctg ctg gac gcc ctg agc cccgag cag cta gtg ctc acc ctc ctg gag 873 Leu Leu Asp Ala Leu Ser Pro GluGln Leu Val Leu Thr Leu Leu Glu 260 265 270 gct gag ccg ccc cat gtg ctgatc agc cgc ccc agt gcg ccc ttc acc 921 Ala Glu Pro Pro His Val Leu IleSer Arg Pro Ser Ala Pro Phe Thr 275 280 285 290 gag gcc tcc atg atg atgtcc ctg acc aag ttg gcc gac aag gag ttg 969 Glu Ala Ser Met Met Met SerLeu Thr Lys Leu Ala Asp Lys Glu Leu 295 300 305 gta cac atg atc agc tgggcc aag aag att ccc ggg atg agg gga aat 1017 Val His Met Ile Ser Trp AlaLys Lys Ile Pro Gly Met Arg Gly Asn 310 315 320 gcg tag aaggaattctggaaatcttt gacatgctcc tggcaactac ttcaaggttt 1073 Ala cgagagttaaaactccaaca caaagaatat ctctgtgtca aggccatgat cctgctcaat 1133 tccagtatgtaccctctggt cacagcgacc caggatgctg acagcagccg gaagctggct 1193 cacttgctgaacgccgtgac cgatgctttg gtttgggtga ttgccaagag cggcatctcc 1253 tcccagcagcaatccatgcg cctggctaac ctcctgatgc tcctgtccca cgtcaggcat 1313 gcgagggcagaaaaggcctc tcaaacactc acctcatttg gaatgaagat ggagactctt 1373 ttgcctgaagcaacgatgga gcagtgaccc tctaatcaac tcggtggcct aaag 1427 16 113000 DNA Homosapiens exonintron junction (465)...(466) exon 1Aintron 1A 16 cagcaccgcgcttttagaat ctcctcagct gaatctgacg ctcagcagtg ggtgaagcgc 60 agccccctgtttcaggccct gccgagctgg aaggagtgtc agagctggag cgcgcgtggc 120 cccctctgtgttggggtcac cccggggttg ccagggctca gggagggtcg tagtctggat 180 tttgtcacccgcacgtcccc accccccagc aggtctgggg ttggagaatc cacgcgggct 240 tcataagctagatgccagtt aactgtcgag aggggacgct ccctcctcgt aggcgtccac 300 actggagaaggaataagatg ggcgattgcc tgggaagcct gacagggcgg cggcagctgg 360 gatgctggagaggactggcc ccttgagtta ctgagtccga tgaatgtgct tgctctgctg 420 gaggaaccgcgctcaggtta cagtcatccc aatatggttc tgaaggtgcg tggttcaggt 480 cacttaggacttgaccagat accgggtttc ttttacaagc cgtttctgac ggtggcctgt 540 ttcaactactggcagagctc atgtaaaaca gacttttaaa aaaatttggg gggcttttag 600 tatttttttcttattcctat attctgagga tattttatag tagtcccaca tatggaatta 660 gataatctcttttttgtttg attaacagtt ttatcaagta taatgtacat accataacgt 720 tcacccattttaatggattc aatgattttt agcatattta cagagtggtg caaccatcag 780 cataatagaattaaggaatc gtgatttttt ttttctggta attgctttta cagttctcaa 840 agtttgcacaagcggatatt ttagaggtac agtgtaatat aagagcttct gaaaatgtcc 900 acttaagttgttttatacct gagcaagtga aattaagaag ggaattgaag caaatattcc 960 tggtaagttgtagggagtga aacttttgtg tcttgtaata ccaagtagat attgaccatt 1020 tcaactggtttttatgctga ggaaatgcat aaaccccatt ttacagatga tgaaatcgac 1080 tttgaaggataagttgccta cagctgcata cctgtgcctg ggctaggccc caaacccaga 1140 tgctttatctctcaatttgt tacccttgct acctcaacag cttggttttc aaccatggta 1200 ctgatgagtgtgaacagtac aagccattca tttactgagc aaataattat tgagtgccac 1260 tctgtgccaagaacactgct ataggtgcta gagatattat tgaatcagat accgtagtga 1320 actgttcctgccctcagctc atcttctggt ggggaggaca atgatcaagt aaagaaatat 1380 atagttttagagattcatct atttttttaa taggtaaatt aaaagggcaa ggaatggcag 1440 tgggaggcagaatctgatga gaaaaatctg aatgaagaga ggaagttagg atataagaaa 1500 gaaagcaagggtttgatttg agcaagcgca aaaatagagt tgtgatttac tgaattgaaa 1560 taaggtgatactggaaggac caggttttgg gggtacaatc ataagtttgg ctttaaatgt 1620 ttttaaataccttgcctctt agacatccaa gtggagatat ggcatttaaa ttcatgagat 1680 tggatgagatcccaccaaag gaacaggttt aggtggagac aaccaaatac cgatgcctag 1740 gacactgcagtgtttagaat tcaaggagat gagaaggaaa caggagggaa gattgaaaag 1800 aagagtccagtgtgttatga ggaaaacccc aagagcatgc tgccttacaa gacaggtgaa 1860 aaatgtgttctgtgaaagaa agagtaatta actgttaaat gttacagact gatcaaataa 1920 aatgaagactgagaatggcc tgtttgtaag gtaataaaaa tacataaaat cttatgatag 1980 aaatatttatacataaagtt agtaaggaaa cagtgtttac tcctttttgt agaagtgtaa 2040 atttttacaaccattttgaa gggcagtttg atattatcta caacttaaaa ttgtgcttcc 2100 attgataatttcacctgtgg aagtttatcc tacaaaaata ttaatatgtg cacacaaata 2160 tgtgtaaaagtgtttatcac agcttgtaca catatatatt tataaatgtg ttgtccagga 2220 acagtggcttatgcctgtaa tcccagcact ctgggaggcc gaggtggatg gatcacctga 2280 ggtcaggagttcgagcccag cctggccaac atggcgaaac cccgtctcta ttaaaaatac 2340 acacacacacacacacacac acacacacac acacacacac acacacacaa attagctggg 2400 cgtggtggcggacgcctgta atcccagcta cttggaaggc tgaggcagga gaatcacttg 2460 aacccgggaggtggaggttg cagtaagccg agatcacgcc actgtacttc tagcctgggt 2520 tacagagtgagacttcatct caaaaaaaaa aaaaaaaaaa aaaaaggtgt ttatcacagc 2580 attgtttacatttgtaaaaa ggtacaagtt ttcatcaaga tggatgcagt tgttaaaggg 2640 aagatataaatgtgtagata tgggagatag ctgctataga cggaattgtg tcccctgaac 2700 tttcatatgttgaagccctt accctgaatg tggtggtatt tggaggcagg gcctttggga 2760 ggtagtttgatttagatgag gtcacgcaga tggggccccc acgatgggag tagtgtcctt 2820 atacaaagaagaagggagtc cagagctttc ttctgtcagt catttaagga catggtgaga 2880 aggcagccatctgtaaatta ggaagagtcc tcaccaggaa ctgaactggc tgtcaccttg 2940 atcttggtctttccaggttc cacagccatg agatatgaat gtctgttttt aaagccactc 3000 agtctgtggtattaatattt tgttatagca gcccaagtta agacagatag ctttgttaaa 3060 tgataaagtcaggttatcta atagaatgca tagtataacc ccatttatct taatgtatca 3120 caggaggcctttctagtcac actaacaaaa gttactcctt tgtgtgcctt ccctgatcac 3180 tgttacattattctatgtac agcacttatt atctaaaatt atttcattaa tttttataca 3240 tgtttactggcttgtcacaa tagaaggtaa gctctgtaag gggtttgcct ctcttgttta 3300 tatccccagtgctaggtata tattacttta ggaaaaacca ttatttatta aaaatatttt 3360 aggaaaaaaccctacacaaa cagtattcct gtagtggttt taaaataaga caacaggctg 3420 ggcgtggtagctcatgcttg taatcccagc actttgggtg gccgaggcag gcggatcacc 3480 tgaggtcaggagttttgaga ccagcttggc caacatggtg aaaccccgtc tctactaaaa 3540 atacaaaagttagcctggcc tggcgtcaca cgcctttaat ctgagctact tgggaggcca 3600 aggcaggagaatcacttgaa cccaggaggc agaagttgca gtgagctgag atcgcaccat 3660 tgcaccgtagtctgggcaac aagagcaaat gtctcaaaaa aataaaataa gaccacaatt 3720 tctttgatagtgtttccttc caaaggtggt ggctaattct cctcttcttg aatgtaggct 3780 ggatttagtgacttgcttct atgtgtagaa tatggccaat gtggaggtat gtcaataggt 3840 catgaattcctttttgttct ctctcttcga tcattcactc tgaagtaaag cagctgcctt 3900 gtcatgagaacatatcaaac agtgctgtgg aaaggcacat ttggtgagaa ataggcctac 3960 tcccaacagccagggaagaa ctgaagcctt ctgtgacatg tgaatgagcc acctgagaaa 4020 tgtatttttcatcctcagtc aatcagtgtc tcaaaagagg ccgttagctg gatccctcaa 4080 caaagccacttttgggttcc tttcagataa tacaggtttg ctttgtaatc tactaggttt 4140 ggtggtagagtgagaagact gaacacactc ccctttagga cacatcataa agcaaaacaa 4200 gtatggcccaaagtagcata cacttaatgt tcttttctac taggatttac agaattcatt 4260 gttggtacaatttactcttt taaaaaataa tttttatgtt gatcagaata aaatacggta 4320 ttccaagctatatgtgctaa cttgatttta ttttaaaaat gtattgaaca ctggaacaca 4380 cagatttgaaagatttgacc ttaatatata tttatatata aaatatgatt ttgaaataat 4440 gaacttttaaatttaaaatt ataaataatt tttaaaatgc cttctattta ggtaaagaat 4500 cttcaaaacaaacttctcat atgatatggt ttgtctgtgt ccccacccaa atctcatctt 4560 gaattgtagctcccataatt cccacatgtt gtgggaggga cccagtggga gataattgaa 4620 tcatggggggtggtttcctc cctgttgttc tcgtcgtagt gaataagtct catgggatgg 4680 ttttattagggatttcccct cttgtttggc tctcattcta ccttgcctgt tgccatgtaa 4740 gatgtatgtttcacctgcca tgattgtgag gcctccccag ccatgtggaa ctgtgagtcc 4800 attaaactttttatttataa attacccagt cttgggcatg tctttatcag cagtgtgaaa 4860 atggactaatacatcataaa agaaatttca ttgcaaaagt tgaagtctga actaaaaagc 4920 tacaaagaaaataatgttta atagccatcc cagatagtgt ccctgaaata cgatgtcaag 4980 gatctagaggaacatattgt atctttaacc agaattaagt ctgaaaaaca agtattcaga 5040 gtcttaaaagaggcaagcag gacttaacgg aacgaattat aaaactaagg tagaaaattc 5100 tagtttatttttgaaacatg tctctcatca taagctcaca tatagcatat gagctccatg 5160 ctcctgattgatcagtttaa tttcatggaa tttcacttat tgcctggtat aacattatta 5220 caatttttcattataagact tgtgattatc aaggtcagga tatcaagacc aacctggcta 5280 acacggtgaaaccccatctc tactaaaaaa tacaaaaaat tagctgggcg tggtggtggg 5340 cacctgtagtcccagctact cgggaggctg aggcaggaca atggcgtgaa cccaggaggc 5400 agagcttgcagtgagctgag atcgcgccac tgccctaccc tccagcctgg gcgacagagc 5460 gagactctgtctcaaaaaaa aaaaaaaaag atttgtgatt atctggtcaa tgtgtgtaga 5520 gaggagatgtttgatcatat acggtaccct tttttttttt tttgagatgg agtctcactc 5580 tgtcccccaggctggagtgc agtggtgcga tctccgctca ctgcaagctc cgcctcctgg 5640 gttcatgtcattctcctgcc ttagcctccc gagtagctgg gactacaggt gcccaccagc 5700 acacctggctaattttttgt gtttttagta gagacggggt ttcaccgtgt tagccaggat 5760 ggtctcgatctcctgacctc gtgatccacc cacctcggcc tcccaaagtg ctgggattac 5820 aggcgtgagccaccgtgcct ggttacagta ccctttttga tagcaggaga aaagatggtc 5880 attaatgtatcctcttataa taagagtaat atttaagaaa gccacaaaat atgaaaagct 5940 tttctatccagatttacatt ctgttgtaga ccatctttat tctgttattt actgtacgtt 6000 agaccaattgatacctttca ttttcctctg gggtttgcat ttcgcagatc acttttaaaa 6060 ggaaaacataggagcctgaa acagaagtgg gaaacaaata tttactcaaa ctaagagact 6120 aaactcagtagccagcaaca agagatcaag gtgtgtgtgt gttttctggt tgtgcagata 6180 ttgtctgaaataagatggct gaaaagttca agtgaaaaag taattaaaag caattcatca 6240 accatagccatagctggatg tataatagct gatcaggcat agcaaactct tcaggataat 6300 ttcatttttaaaaatttatg tctttgtcct tttcatcttc taagcacagt ttcaaataag 6360 actacagagtgaggctctag ggaccatcag tttttgtctt tagtgctaaa atggtggctg 6420 agtgacacaccatgattttt tttctcaata tttcatcatt ctaccagtgt tggaaaaggg 6480 agagaaggactctctgaagg agactgtgca aaggattctt tttttttttt tttttttttt 6540 gagatggagtctcactctgt tgcccgggct ggagtgcaat ggcatgatct cggctcatgc 6600 aacctccacctccctggttc aagggattct cttgccttag cctctttagt agctgggatt 6660 acaggcgcgccaccacgctc ggctaatttc ttgtattttt agtagagaaa ggatgtcacc 6720 atgttggtcaggccagtctc gaactcctga cctcgtgatc tgcccacctc ggcctccgaa 6780 agtgctgggattaccagcgt gagccactgg gcccggcccc aaaggatctt tttacaccat 6840 gtctggttcccagccctttt tctatccttc ctgtgcagtg tggactgagt tgactgagat 6900 atttaggcccaggacttctt gcttgttcta tagttattga gaaaagtgtg tcaaaatatc 6960 catcactgattaaggatttg tctgtttatt tagttctatc aacatttatt ttttaacttt 7020 gaagctatttgcatacaaat tgaggatttt tatctttcta ttgaattgcc ccttttatcg 7080 ttatgaaatctcacttattt catgtaatac tttttgccct atagtctagg ttgtctgata 7140 ttaacatagctagataatat ttcttagatt gcatggtatg tatttttcca tttttcattt 7200 tcaatctttctatgtgatta aagtatgtct tttgtaaaca gcatatagtt ttgtttttta 7260 atctagtcttataatctttg tcttttaatt ggaatgttta ggctatttac attaaattct 7320 gatattgttggatttaagtc caccatactg ctacttactg tgttttttct cctctggtct 7380 ttgttcttgtaataattagt ttgttttttg ttattgttga tttttttttt tttttgtcaa 7440 gatggagtcctcctctgtca cccaggctgg aacgcagtgg tatgatctcg gctaactgca 7500 acctcagcctgccaggttca agcaattctt ctgcctcagc ctcccgagta gctgggatta 7560 caggtgcctgctgccatgat gattaatttt atgtgttaac ttagctgggc tgtgttgccc 7620 agatagttggttaaacatta ttctggatgt ttctgtgaag atgtttttgg atgaggttaa 7680 catttagatcggtggacttt gagtaaagca gattaccttt cataatttgg gtggggctca 7740 tccaatcagttgaacatctg aagagaccaa aagactgacc ttctgcaagc aaagaaaaat 7800 tctgccaacagacagccatt ggacttgaac ttcaacattg actcttcagt ctattggccc 7860 accctgcaaattttggactt gccagtaagt gtctgaaatc tagtgaggca atttctttct 7920 tttttttttttttgagatgg agtttcgctc ttgttgtcca ggctggagtg cagtggtgcg 7980 atctcagctcaccgtaacct ctgcctccca ggttcaagtg attcttctgc ctcagcctcc 8040 tgagtagctgggattacagg catgtgccac cacgcctggc tacttttgta tttttagtag 8100 agatggggtttctccatatt ggtcaggctg gtctcaaatt cccaaactca ggtgatccac 8160 ccgccttggcctcccaaagt gctgggatta caggtgtgag ccacagtgcc cagcctaatt 8220 tctttctttctttctttctt tttttgagac agagttttgc tctttttgac cagaaaggag 8280 tgcaatgtggcaggatgttg gctcactgca acctccacct cctggcctct ctagtagctg 8340 ggattacaggcgcctgccac cacgcccagc taatttttgt atttttagta gagatggggt 8400 ttcaccatgttggccaggct ggtctcaaac tcctgaaatt acgtgatctg cccgccttgg 8460 cctcccaaagtgctgggatt acaggcgtga gccaccatgc ctagccgggt agtttatctt 8520 gacttgacttcaggctcacc aatccttttg gctgcaattc tacgatagaa aaggacataa 8580 aaaactttaaattagcctta gaataaagag atgttatcat tccctagcaa ttagtattca 8640 aagcaagatccaaatatgta attagtcatt tatgtatcta agctgtttgt atgtatgata 8700 caagttttcacatacaaatt tcttctttct ttctttcttt cttttttttt gatagaggca 8760 gggtttcacgacattgccca ggctggtctt gagctcaagt gatccatctg ccttggcctc 8820 ccaaagtgctgagattacag gcatgagcca cagtgcctgg cccaaattat tgtagttatt 8880 tccaattcctttcccccttc tcacatccca attaaagaat tccactcagg aattgttgta 8940 gtagaagtgctttagtctgt gtgctacggt ttggatactg tttgtttgcc aagtctcatg 9000 ttggaatttgatcactaatg ttgaaggtgg agcctggtgg gaagtgtttg ggttgttaag 9060 gcagatcccttatgaatggt gtggtgccct tctagaggga gtaagttcgt tctcactctt 9120 ggttcccacaagatctcgtt gttgtaaaga tccttgtact tacccctcct ctctctcttg 9180 ccttctctttcaccatgtga tctacacaca cagtatcata aggcatcttt ctgatccttt 9240 agtgttcactctccagtacc tttaatattt gccttcaaat ttctcaaatt tctttattta 9300 cttccatttttctcctacaa taattgtagg cgtacttaaa gtagaattac aatataaata 9360 atattttaaaatatctacaa ctaatactaa aggggttact ttattttatt taaattttat 9420 ttttaaataagaatttaaaa tatctgcaac taatatcaga gccaaggggc tactttcttt 9480 gaaatacaaagagtctttag agtcagactg tgtatgtttc aatctgggat ctacctctta 9540 tattgtaggtttagacaaat tgctaaatat ttcttgtccc agttttctca tctacaaaat 9600 ggaaaaattagcttcccttt gctgtctgcc ttgagtagaa gcttcctgag gccctcatcc 9660 aaaacagatgttggtgccat gcttctagta cagtctgcag aactgtgagc caaataaacc 9720 tcttttctttataaattact cagcctcaag tattccctta tagcaacaca aatggactga 9780 gataccgtgtgtgatgtcct aatccttata atattatcct actacccagg cagatattgc 9840 tctccaaatgtcttcttaaa aaggatggtt tctgaaatga caccctcttg ggactattgg 9900 aattactgaacagctgtttt cattagaaat cttttttttt ttttgagaca gggtcttgct 9960 ctgtcgcccatgctggagtg cagtggtgca atttcagctc actgcaacct ctgcctccca 10020 ggttcaagtgattctcctgt cttggcctcc tgagtacctg ggactacagg tgtgcaacac 10080 cacacccagctaatttttgt gtttttagta gagatgggtt tcattattta tttatttttt 10140 tgagacgaagtctcgttgtg tcacccaagc tggagtgcag tggcgtgatc tcggctcact 10200 gcaacctccacctcccaggt tcaagtgatt ctcctgcctc agcctcctga gtaactggga 10260 ctacaggtgcacaccactat gcctggctaa tttttttttt tttttttttt tgtattttta 10320 gtagagacaggtttcaccat gttagccagg ctggtctcaa actcctgatc tcaggagatc 10380 cacccgctttgcccttccaa agtgctggga ttataggctt gagccactgt gcccggcctt 10440 agaaatatattttgactata catatatttt gtttttattt atttttattt ttttgagatg 10500 gaggcttgctctgttgccca ggctggagtg cagtggtgtg atctcagctc actgcaacct 10560 ctgcctcctgggttcaagtg actctcctcc ctcagcctct gaagtagctg gaattatggg 10620 cacatgccaccatacccagc taatttttgt gtttgtattt gtatttttga ggtggggtct 10680 tgctctgtcgcccaggctgg agtgtgtggc attatcttgg ctcactgcaa cctccgcctc 10740 ctgggttcaagcaattctcc tgcctcagtc tcccgagtag ctgggattac aggagcccgt 10800 caccacacccggctaatttt tgtagtttta gtagagacgg ggtttcacca tgttggccag 10860 gctgatctcgaactcctgac atcaggtgat ctgcccactt tggcctccca aagtgctggg 10920 atgacaggcgtgagccactg tgcctggcct aattttgtat ttttagtaga gatggggttt 10980 caccatgttggccaggctgg tctcgaattc ctgagctcag gtgattcacc tgcctcagcc 11040 tcccaaagtgctgggattac aggcgtttgc cactgtgcct ggccaactat atatatattt 11100 taaaaggggacatttctttt taattttgga atggacattt gaaaattgtt tgaattactt 11160 tagtctactcatatctttca gtctattgac acaaggtata tctggtttaa agagaaaagg 11220 tggaacaaaaaaaacccatt ctagatcaat tggtagatgc caacagattc actcccatat 11280 gaatatgaaaggacaaggaa ccatgaatat tttcatgatg aaggtgagaa taagttttga 11340 ttgatttttgaagaaaaaca atttttgtta tcttgtttaa ctctaggagg taatcgagaa 11400 atgttgagttgtttgttggt tctctcccaa agggagggta gaaggaagcc atggttcctt 11460 tataccgtggttgactggga gcctttatgc ctttctgata tattaagaga aaatgcaagg 11520 ggggcctaaaggtctctgtg atactgaaga gaaaggtata ggggtaatag ggctgtgaga 11580 aagctgaaagctgagatcat gttacagaat aagatagcgg agtttcatat ttctggtatg 11640 gggcaattcctgctgatgac aaaatccagg gttgtttttg gatctaggtg taggtggttg 11700 aagtagggtataaaggcagt catgtgctgg taaactggct cttgagaaaa agcacccaat 11760 ttgagcattcattgactttt gataccaaca tgtcattgag catagaatta gaaagagata 11820 tgaataatcaactcttggga gctggaatga tctggcttta acaaccactt tctacatcaa 11880 aaaaagttaatgttattaat attagaataa taaataatta aataataaat gagtgtaggt 11940 gtagggcattggaattaagt acacacatga atcacaaagc tgtattattg gatcgatcat 12000 ctactgtgacccctgaaatc ttgaattatg gtatgagttg gtatagaaga agaatgtgag 12060 gccccaaatcttcattgagt gaaggagggt tgaggagtag tcagtagaaa agaataaaaa 12120 gagaagattttatagaagtc tgttgggggt aaaatattgc tgaggaagta aaatagtact 12180 gaggaagtgttcttcaaatt ccttcgacta taaccacttt ttaatgtaat ctgtatgtaa 12240 agcaagggtctacatgatcc aatttatgtg ttggctccat ttataaaaga atatttcagt 12300 tgtcaaaactagttgacagt acagttaatc ctttaataat ctggggttag gggatatggc 12360 catcatgcaaaaaaaaaaaa aatctgtgta taattgttga ctcctcccaa acttaactac 12420 taataacctgttgttgactg gaagacttac cagtaatata aacagttgac taacacatat 12480 tttgtatgttgtatgtatta tacactgtat tcttacagta aagctagaga aaagaaaatg 12540 ttattaagaaaatcataaag aagaaaaaat atatttacta atcattaagt ggaagtggat 12600 catcataaaggtcttcattc tcatcgtctt cactttgagt agtctgagaa ggaggaagga 12660 aaggaggggttggtcttgct gtctccgggg tagcagaagt agaagaaaat ccacgttatc 12720 agtggacccatgcaattcaa atcggtcttc aagggtcaac tgtaattcca atcttaatta 12780 tttgccttaactaattttct taataaaagg tggaatattc ataatttaca ataacacctt 12840 cattttcttaacttttctca ctatatctct cacatcacat cctaaacctt tttctcctgt 12900 gcctaactctccattctctt aaaaaactct cccagatcca gtctatgctg ctcataattt 12960 ctcttcccttcctctttttc ctaccttctt tctaatgcaa attcatcatt tcatgaataa 13020 ttttctctcctcttatttcc tacttttact caacaaaagt ccagaaacta aacttgctta 13080 ctcagatcccagagctgcat aaaaggacag gagatcttgg atgatgtgtg ggttggaaac 13140 agaaggtattacattctttt gttaaataat tgaggatttt gcatgtggtt aaaatgatgt 13200 cagagctaggcaaggaaacg ggattctcct acattcctga taggagatta aattggtaca 13260 acccatttggaaatgcattt gtcaatatct cctaaaacca aagtgtatcc ctaaaaccag 13320 aatatatcctaccctgtgac tcagcaattc cactccatca acagtggaat gtaatgaata 13380 tggctatcaggtttcaatat gctagtgaca tctgctacat ctattaacag aagtctataa 13440 ttttttaacctctgatctct gaaaacttat tttatgactt tattactcta caaactaaaa 13500 tgtcttactattgtgtatca gatccacttc tttttaaatt aatttttaaa tgtcaagtct 13560 taatagtcttcctttagcct ctatttacta atttattgtc cccacaatgt cactctaaac 13620 gtaactgttaactatcagga agtatttcct tctttttcta tagagaacag aagatcttca 13680 gcaggaaattcagatgctta ctcagcaaat ggaacagctg tatcatcttt atgaacagct 13740 gtttgtgaatcattccaact tgaagaaaag tatagggaac aacaaaagat ccttgaaata 13800 cctggaaggaaaaattgctt ttaatgatgt tttaaaagat tagactatga aaaaagactt 13860 tcctgaattatagatgttat tttgggcaat gaaattaact atttattatt ctaatattaa 13920 taactttaactttatttggt gtagaaagct gataaaaact atttatattt cacttaacat 13980 tggaaaagtgagggggaaaa atccttagag ttatgcttct aattttatca aaaaacatgc 14040 cctttcccatatcttcagtt ttttcactgt gtacatattt gacagataaa accatcaata 14100 taatatggaaagtttagtgt cttttataat ctcttcttgt aagttacata acatcacttc 14160 tgccatattctatcagtcaa acagaccaac ctgatacaat gttgttcgag actataaggg 14220 taccaggcggcgcggtgcag atcattggtg gtcatcttaa aagtctggct accgcactca 14280 tttcttttactcacaaactg aatgatttgc ttattcattc atttattcaa tacttattga 14340 acaaccacagattttacaaa catataggac tggctggggg cagtggctca cgcctgtaat 14400 cccagcactttgggaggctg aggtgggctg atcacctgag gtcgggagtt caagaccagc 14460 ctggccaacatggagaaacc ctgtctctac taaaaacaca aaaattagcc aggtgtagtg 14520 gctcacatttgtaatcccaa ctacttggga ggttgaagca ggagaatcga ttgaacctgg 14580 gaagtggaggttgcagtgag ctgagatcgt gccactgcac tccagcttag gcgacagagc 14640 aagactccatctcaaaacaa acaaacaaaa taggacttaa tgaagtaagg tcaacagtag 14700 accatatagagtttaaagat aaatatatca tctcatctag cctccacctc tgcctttgaa 14760 tatgtgtatggaaataatac attgaatggt taatccatgc aaataaaaat aatcctttat 14820 taagttttcttaagattgta caaaataacg tgtgcttggc caggcatggt ggctcacacc 14880 tgtaatctcaacactctgtg aggccgaggt gggatcactc gaggtcagga gttttaagac 14940 cagcctggccaacatggtga agctctgtct ctactaaaaa cacaaaaatt acctgggcac 15000 ggtagcacatgcctgtggtc ccagctacct gggaggctga ggtgggagaa tcatttgaaa 15060 ctgggaggcagaggttgcag tgagccaaga ttgcaccact gcactccagc ctgggcaaca 15120 gagtgagaccctgtctcaaa aaacaaacaa aaaacaaaat aacatgtgcc taccccaaca 15180 cttaaagctatgctaaacag tttaaaggaa ataataattt tctctctgcc catgtcacct 15240 cagtaaccaatgttaaccat tcccatagtt atggaaatat gtaaacatat ataaagggta 15300 atggtgtcttcacaaaacta agatcattct aataaaaata ttctgcaact tcctctactt 15360 agtagtgcctcatggttgta tcttaagtta aaagatatag ctcttccttt aataactgta 15420 taatattctatagtatgcat gtatcttaat ttattcaacc atttctcttt tgagggatga 15480 tataattatttccttctttt ggtcactaca aataatgtga aaataagtat ctttcaactt 15540 atatccttccacactggtgc ttttgttgct aggggattaa ttgacaaata tgagctgata 15600 gggtcacagtgcgtatttta aattctaata gccattgtca gattactatt tgcaaaagga 15660 tagaagcagttcatttaaga gtaaatcatt ctcctttaca tccagctagc attgaatgct 15720 gtcattcttttttgttgtta gttgggtaaa aaaagaaaca aaaaacaagg tacctcatta 15780 ttattgtaatttacattttc ttgactacta gtgaagataa ggatcttttt tttttttttt 15840 ttttcctttctgtggagata aggtcttact atgttaccca gactggtctc aaacccctgg 15900 atcaagctatcctcctttct cagcctccca aagggctgaa attacaggtg tgagtcattg 15960 cacttagccagtaagcatcc ctcttcttta aaaaaataat ttcaggccag gtgcagtggc 16020 acatgcctgtaatcccagca ctttgggagg tcaaggtggg tggatcacct gaggtcagga 16080 gttcgagaccagcctggcca agatggcaaa accctgtctc taccaaaaat acaaaaatta 16140 gctgggcatggtggtgggta cctgtaatcc cagctactcg ggagcatgag gcaggagaat 16200 ggcttgaacccaggaggcgg aggttgcagt gagctgagat catgctattg cactccagcc 16260 tgggtgacaagagcaaaact ctgtctcaaa taataataat aataattttt atttttatta 16320 tagattaaggggtacatgtg caggtttgtt acatgggcat aatgcgtgat gctgaggttt 16380 gggttacgtcaccaggtaat gagcttagta cccaataggt gattttgcat cccatgtccc 16440 ctctctcccatgtctggtag tccccagtgt ctattgttcc cacctttatg tttatgtgta 16500 ttcaatgtttagctcccact tataagtgag aacatgtggt atttggcttt ctgttcttgt 16560 gttaatctgcttaggataat ggctgccagt tccatctatg ttgctgcaaa ggatgtgatc 16620 tcattctttttaatggctgg taagcatctt catatatgcc tgttgaccac tgggcttttc 16680 ttttctacaaattgcctcct tcttcccata atttggatct taggtgcaga agattgtgct 16740 aatcaaatttcttaaatagt gtcttgtcat tggggacata atggtccatc tctatttaat 16800 tttattgtttttggttccat tccccacttc cattccttat gcccataggt agcctcactt 16860 aaatgtgtttatgtctatca ttttgtttat gtgattaaaa aatcattatt gggatattta 16920 catgccataaaattcactca tttaaagtct acaattcaat gatttttagt aagttaataa 16980 agttgtgcaaatgccaccac aatccaggtt tagaacattt ccatcaccca aaaaagattt 17040 ttttttttttttgcttctag acaattaatg ccctcttcca tcactagtgc cgggcaacca 17100 ccaatctgctttctgtgtgt atacattttc ctttttttgg acatttcata gaaataaata 17160 actttaatatgtagtctttt gcatctagtt tttaaaatta gcattgtttt tgaggtccat 17220 ctatgttgtagcattcatca gtattgtgtt ctttttatta tttaatggta ttctattgtg 17280 tggatatgccacattaaaaa ataatacttt atttttggaa gcaattatag ggttacagaa 17340 aaattgactataaagtacag agatcccata aacttccttc cccatcttca cagtaacaaa 17400 ttgcattagtgtggtaaatt tgttacaatt gagttaacat taatacatta ttattattat 17460 tattattgaggcggagtttc gctcttgtta cctaggctgg agtgcaatgg catgatctca 17520 gctcactgcaacctccgcct cctgggttca aaagattctc ctgcctcagc ctcctgagta 17580 gctgggattacacacatgca ccaccacacc cgactaattt tgtacttttt ttagtagaga 17640 caggatttcaccatgttggt caggctggtc ttgaactgct gacctcaggt gatccgcctg 17700 cctcagcctcccaaagtgtt gggattacag gcatgagtca ctgcgcccag cctgatacat 17760 tattattaactaaagtccgg ggtttacatt aggattcatt ctgtaatgta cattctatgg 17820 gttttgaaaagtgtataatt acaagtatcc atcattacat catcatacag aatggtttca 17880 ctgccctaaaaatgtcctgt gttccatctg ttcattcctt cctcctcctg caaacctctg 17940 gcaaccacaacttttttttt ttgagatgga tgtctcgcta tgttgcccag gcttatctca 18000 aactcctgggctaaagcaat tctcctgcct tagcctcctg agtagctggg actacaggtg 18060 tatgccaccatgcccggctt gatcttttta ctacctccgt agttttgtct tttccagaat 18120 gtcgtgtatttggaatcata cagatataac cttttcagat tggcttcttt cacttagtaa 18180 tatgcattaaagttttctcc atgtcttttg gtggcttaat agctcattgc tttttattgc 18240 aatgtgaataaaaagcattt tttttttgca aataatattc tgttgtgcag acttactaca 18300 ttttagctttccatttacct aatggtaaat cttcgttgct tccaattttt gacaattata 18360 aataaagctgctataagcat tcaagtgcag gtttttatgt ggacataaat tttcacttca 18420 cctgggtaaaaaccaaggag tactatttct gagtcttatt gtaagaatat gtttagtttt 18480 gttagaaactgccaaactgt tttacaaaat ggctgttcca ttttgcattt ccatcagcaa 18540 tgaatgagagctattgctgt cactctcaca tcctcaccag catttggtgt tgtcagtgtt 18600 ctggattttagccatttgaa taggtgtgta gtggtatctc atcattgttt taattgcagt 18660 tccctaatgacatatgatgt tgaacatctt ttcatatgct tatttgccat ctgtatatct 18720 tctttgatgagaacttttgt tcagaacttt tgccattttt aaattgagtt ctttattttc 18780 tagttgttgaattttaaatt ttatttgtat attttgggat aacaatcctt tatcagatat 18840 atcttttgcaacaattatct cccagtctgt ggcttgtctt ttttattttc ttaatagtct 18900 ctatcacagggcatactttt tagttttaat gaagtccaac ttgtcagttt tttttttcat 18960 gaatcttgcttttctattgt atccaaaaaa tcatctctaa acctaggtca cttacatttt 19020 ctcctacgttgtcttctagg agttttatag ttttgtactt tacatttagg tctgtgatgt 19080 attttgagttagtttttgtg aaggtggtat gaggtctgtg tctggattca ttttttgtta 19140 atgtggatatgtagttgtat gtagttgttc tagtaccatg tgttgaaaag actatccttt 19200 cttgattgaattgccttgtt cctttgttaa agatcagact ttggatgagt ctatttcttt 19260 aatttctttcatccaagttt taaaatagtc ctcatttaga cttttttttt ttttgagact 19320 gggtctctctctttcaccag ggctggaggg ctggagtgca gtgatgcaat cacagctcac 19380 tgcagccttgacctcctggg ctcaagtgat cctcccatct cagcctccct agtagctggg 19440 attacaggcacatgccaacc acgcctggct aattgtattt tttgtagaga taggattgca 19500 ccatgttgcccaggctggcc ttgaactctt gggcttaagc aatctgcctg ccttggcctg 19560 ccaaagtgctgggattacag gcatgaacca caacacctgg ctagctaatt taaaattttt 19620 tcttttgtagagatggaatc ttgctgtgtt gacctggcta gtttctaatt cctggcctca 19680 aatgatcctcccaccatggc ctcctggggt gctgggatta cagatgtgag ccaccacacc 19740 cagcatattttgttagattt atacctaagt atttaacttg cttgataatt taaatttttt 19800 tttttttttttttttttttt tttttgagat ggagtttcgt tcttgtcgcc caggctagag 19860 tgtggtggcacgatcttggc tcactgcaac ttttgcatcc cagattcaaa ggatgctcct 19920 gcctaagcctcccaagtagc tgggattaca ggcatgtgcc accatgcctg gctaattttt 19980 gtatttttagtagagacagg gttttactat gttggtcagg ctggtctcga actcctaacc 20040 tcaagtgatccacctgcctt ggcctcccaa agtgctggga ttacaggcat gaaccaccgc 20100 acccggccgatactttaaat gttattgtgc ttttaatttc aatttctaat tgttcatatt 20160 tggtatattaggaaagcaat tgactttgta tattaacttt gtattttgca accttgctgt 20220 aattgtttattagttccaga aattttaaaa gtcaattctt tgggattctc tacatagaga 20280 atcatgtaatctgtgaacaa aaacagtttc atttcttcct tttcaatctg tattaatttt 20340 cttttcttttcttgcctcat tgcactggct agatcttcta gcattgtact gaataagaac 20400 aataagcatggatatcctgt tttcaatctt agagggaaag cattcagtct ttcaccatta 20460 aatgtaatgttaaatataga tttttttata gatgcttgtt atcaagttga gaaagctccc 20520 ctgtattcctgtttttctga gtttattttt atgagtggtg ttgaattttg tcatgctttt 20580 tctgtgtctattgatatgat catatgtttt tcttttctag cctgttaaca tagtgagtta 20640 cattgatttttgaaggttga accacccttg catctctgga attaaggcct gatattgttt 20700 ggatatttatgctacccaat tctcatggtg aaatgtaatc tccattgttg gaagtgtggc 20760 ctggtgagaggtgtttgggt tatgggggca gatccctcat ggcttggtgc tgtcctcacg 20820 atagtgagtgagttctcacg agatctggtt aatttaaaag tgtgtggctc cctccctgtc 20880 tctctatcttgcttctgctc tagttatgtg atatgctgtc aggtgctggg ctcccccttc 20940 accttctgccatgattgtga gcttcctgag gcctcactgg aagctgagca gatgccccgc 21000 accatgcttcctgtacagcc tgtagaacta tgagacaatt aaacctattt tctttgtaaa 21060 ttatccagtctcaagtattt tttgtttgtt tgtgttgtga gatagggtct cactctgtcg 21120 cctaggctgtagtgcagtgg tgcgacctgg gcccactgca acctctgcct ctgggttcaa 21180 gtggttctcccacctcagcc tcctgagtag ctggaactac aggtgtgtgc caccacaccc 21240 ggctaatttttgtatttttt ggtagacatg gggtttcacc atgttggtca ggttggtctt 21300 gaactcctgacctcaagtga tcagcctgcc ttggcctccc aaagtgctgg gattaccagc 21360 atgagccaccacagttggcc tcaagtattt ctttatagca atgaaagaat ggccaaatac 21420 aaccccactttatcatggta tataattcct tgtatgtatt gctgaatttg atttgataat 21480 attttgttaaggatttttgt atatattcat gtggtatatt agtctgtagt tattttattt 21540 tatttttattttttgagatg gagtcttagt ccattgccca ggctggagtg cagtcgtggg 21600 atctgggctcactgtaactt ccaccttctg ggttcaagtg attctcttgc ctcagcctac 21660 aaagtagctggtaccacagg tgcgtgccac catgcctgac taatttttgt atttttagta 21720 gagacagggtttcaccatgt tggccaggct ggtctcaaac tcctgacctc aagtgatcca 21780 cccaccttggcctcccaaag tgctgggatt acaggcaaga gccaccgtgc ctggccacag 21840 ttatatttttttggattgtc tttgtttggt ttttatatca gggtaatatt agtttcataa 21900 aatgaatttagaagtattct ctgtgtctat tttttggaag atattgtgta ggattagtgt 21960 taactcttcttttaagattt gatagaattc tccagtgaga ccatccggat atggagattt 22020 ctgttatgggaagttttaaa attataaatt ctggctgggc actgtggctc atgcagtaat 22080 cccagcacgttgggaggctg aggcaggagg atcacttgag cccaggagtt tgagaccagc 22140 ctgggcaatagagtgagacc ctgtctctac agaaaaaaaa aaaaaattag ctgggcatgg 22200 tggcatgtgcctatagtctt agctactcga gaagctgagg tgggaagatg tcttgagcct 22260 aggagttcaaagctacaatg agctatgatc atgctgctgc actccagcct gggtgacagt 22320 gagacactgcctctaaaaaa taaaaaagta aaaataaatt ataaattcaa tctctttaat 22380 agttaagggcaattaagatt atctgcttaa ggccaggcgt ggtggcacat gcctgtaatc 22440 ccagcactctgggaggctga ggcgggtgga tcacgaggtc aagagatgga gaccatcctg 22500 gccaacatggtgaaacactc tctctactaa aaatacaaaa attagctggg cgtggtggca 22560 cgcacctgtagttctagcta ctcaggaggc tgaggaagga gaattgcttg aacctgggag 22620 gcagaggttgcagtgagctg agatcatgcc actgcactcc agcctgtcaa cagagcaaga 22680 ctccatctcaaaaaaaaaaa aaatacaaaa aatacaaaaa attagccagg tgtggtggtg 22740 cgtgcctgtagtcccagcta ctcaggaggc tgagacagga gaatcgcttg aacctgggag 22800 gcagaggttgcagtgagtca agatggcgcc actgcactcc agcctgggca acagagcgag 22860 actctgtctcaaaaaaataa ataaataaaa cattaaaaaa agataaccta cttaatattg 22920 gatgattgtagtagtttgtg tttttcaaag aattggttca tttaatgtaa attgtccagt 22980 ttatgtgtgtagagttgttt ataataattc cttattattt tttagacatc tgtatagtct 23040 gtagtaatagaccttgcatt ctgaatactg gtaactagcg tcttctctct ctctcctttt 23100 tttttttttttttttttttg agacagactc tcgctctgtt gcccaagctg gagtgcagtg 23160 gtgcgatcttggcttaccac aacctccacc tcccaggttc aagtgatttt cctgcctcag 23220 cctcccgagtagctgggact acaggcacac accaccatgc ccagctaatt tttgtatttt 23280 tagtagagatggggtttcac tatgttgcca ggctggtctt gaactcctaa ccttgagatc 23340 tgcccgccttggcctcccag agtgctggga ttacaggcat gagccaccgc gtccatccag 23400 tcttctctcatttgtgcttt gttagtcttg atagaagttt gtcaatttta ttaatttttc 23460 tttttttttttttttttttt tttttgagac ggagtcttgc tctgttaccc aggccagagt 23520 gcagtagtgtgatctcggct cactgcaacc tccgcctccc aggttcaagt gattctcctg 23580 catcagcctcccgagtagct ggaactacag gcttgcacca ccaggcccag ctaatttttg 23640 tatttttagtagagatggag tttcgccatg ttggccaggc tggtcttgaa ctcctgacct 23700 caggtgatctgcctgcctca ggctctcaaa gtgctgggat tacaggtgtg agccaccgtg 23760 cccagccgattttattaatt tttcaaaaga accagttctt tgtttcattg gtttttctat 23820 ttttttcctgttttacattt aatcaatttt gttcttattt ttattatttc cttccttctg 23880 cttgctttggatttattttg ttcttatttt cctaggttct tggtgtggga gcatagatta 23940 ttaatttgagatcttccctc ttttctaata cacacattta gtgctataaa tttccctctt 24000 ggtggtgctttagctgtgtc cctcaagtgt tgatatgttt tattttcatt ttcattcagt 24060 tccatgtatttttaaaattt cccttgacct atgttttatt taggagtact tgtttcattt 24120 ccatgtgattggagattttc ctgttatctg ttattggttt ctagtttgat tccactgtgg 24180 tcagaaatcacattctatac gatttcaatt cttgtaaata ttttgatgtt tgttttaatg 24240 ctcaggatatggtctatctt actatttctt gcatagaccc tcaaaaggtt gtgtagcctg 24300 ctcttgtagggtggagtatt ctacaaatgt caattggatt ttgttgatgc tggtgtggtt 24360 gagtttttctatgttcgtgc tgattatcta tctcattcta tcaactgaga gaggagctga 24420 atcctccaacaatagtggat ttttctcttt cttctttctt tctttctttc tttttttttt 24480 ttttgagacagagtctccct gtgttgccct ggctggagtg aagtggcgag atctccactc 24540 actgcaagctccacctcctg ggttcatgcc attctcctac ctcagcctcc cgagtagctg 24600 ggactacaggcacccgccac cacgcctggc taattttttt tgtatttttg gtagaggtgg 24660 ggtgccaggatggtctcgat ctcctgacct tgtgatccac ccgcctcagc cttccaaagt 24720 gttgggattacaggcgtgag ccaccgcgcc tggcctcttc tttctttttt cttttctttc 24780 tttctttctctttctctctc tctttccttt tcttttcttt tttttttttt ttgacagagc 24840 ctcactgttgccccaggctg gagtgcagtg gcctgatctc ggctcactgg aacctccgcc 24900 tcccaggttcaagtgattct cttgcctcag cctccagagt agctgagact acaggtgtgc 24960 accaccacatctggctgatt tttgtatttt ttattagaga tggggttttg ccatgttggc 25020 caggctgctttcaatctcct gacctcaggt gatacacccg ccttggcctc ccaaaatgct 25080 gggattataggcatgagcta tcatgcctga ccttttttct ttcatttcta tcagtttttg 25140 cttcacatatcttataactt tgttgtttgg gggcatttaa gattactgtg tcttcttggt 25200 tgattgatccttttgttatt atataatgtc cctccctgtg tctggtaatt ttatttgctc 25260 tgaagtctactttgtttgac actttccttt aatatttgca taacatattt tttccatcct 25320 cttactgtcaaattccttat atttttattt gaagagtttc ttatagatac catatagtta 25380 aacatcttttaaatcccctc tgctaactct gtcttttaac tggggtattt atttttattt 25440 atttttttctttttgtgatg gagtctcact ctgttcccca ggctgtagtg tagtgatgct 25500 cacttggctcactgcaacct ctgcctcccg ggttcaagtg attctcctgc cttggcctcc 25560 caagtagctggaattgcaga tgtgcaccac catgcctgga taattttttt gtatttttag 25620 tagagactggccaggctggt cttgaacttt tgactgtatg ggaacagaca cacaactctc 25680 ccaaataagcacaacaaaga gacacagaag cagtccaagc ctctgataaa ctctcccatc 25740 ctgaatccttaaaaatgctt agtctgtaag aggatgtgcc tctgacctaa ctcagccaga 25800 cgcccctctcaggtttgttt tttctaaaat aaacctgtct tgactggcaa gccacctttc 25860 ttttctctcctctttcttta attcctacac tgacttcaag tgatctgctt gcttcggcct 25920 cccaaagtgctgggattaca ggtgtgagac actgcgcccg gcctaactgg tgtatctaga 25980 ccatttacatttaatgtaat tattgctata ttagggctta agtcttcctt ttcattttgt 26040 tttctctgttttttaaattt ctgttttctt tttcctaatt tcatgcttgt tcctgaaaca 26100 ttttttagaattccattttg aattatttat agtttttgat gataaacata tatatttggt 26160 atagcttttttagtggttgc tccaggtatt acattttgta tatatgactt aatacagtgt 26220 attgatgtcattttaccagt ttgagtaaag tatagaactc ttagcttcca ttatgtctct 26280 acttttccctgtttatataa ttatcttagc tatttcctct tcatacattt agaaccacat 26340 catacagtgttatagttttt gctttaacca tcaaacatat tttagaaaac tcaagagaag 26400 gaaagcctattgtatttacc cacagttttg ctcattatat tttctgtctc ctgatgttcc 26460 aagattccttcatttttaaa aatcattttc tttctgtttg gagaacttca ttatttagta 26520 agtctttttgtttttgtttt tgtttttttt tagagatggg gtattgctgt cacctaggct 26580 ggagtgcagtagtgtgatca tagctcactg cagccttgaa ctcttgagct caagcaatcc 26640 ccctgctcagcctaccaaat agctggtact acaggcatgc accaccatgc ctggctaatt 26700 ttttttttttttttttttct gagatggagt ctccctctgt cacccaggct ggagtgcaat 26760 ggcgtgatctcagctcactg caacctctgc ctcccaggtt caagcaatta ttctctcatc 26820 tctgcctcctgagtagctgg gactacaggc acacaccacc acacctggct catttttgta 26880 tttttagtagagacagggta tcaccatgtt ggccaggctg gtctcaaact cctgacctct 26940 agtgatccgcctgcctcagc ctcccaaagt gctgggatta caggcgtgaa ccaccatccc 27000 cagtgtgttgtaggctttta aaatgtaaag caaaattgtt ctaccagcag tgaatcaaac 27060 agtaggttttgaaacgtcaa gaagcccaaa cacaaattta agttagagtt ttgtaaagta 27120 atataagttctcctttaaat gcattttaaa atattaataa ttttctttag tattgcttaa 27180 ccccctgtaagtcactaggg ctccataatt attttggaac caactcctaa gttaatattc 27240 tttcactgtaatttcagcat ccttaaatct tctaagcaca gctataagtt gaaatgattt 27300 tagagaactgtgagtaaaaa tctaatatga taaaatggct ccattttgcg gggaaggatg 27360 tactggtaattgacagaaaa tgaccaggaa catggaaata ggagtaggtc agacagattg 27420 aattgttaagtattttgaat atactataaa tgagatataa atgatatttt gaaatcaata 27480 tgcaatttttgttgtatcta ataaggactt ttaaggatac agtcaagaag gagagatgca 27540 atattactgtgtttagcctt actaaagcaa aggaaagtac tgtacgtaaa agttctctgg 27600 cgcggtggctcatgcctgta atcccagcac tttgggaggc cgaggcgggc agatcacgag 27660 gtcaggagttccagaccagc ctggccaaca taatgaaacc tcgtctctac taaaaataca 27720 aaaattagttgggcgtggtg gtgtgcacct gtaattccag ctgcttggga ggcagaggca 27780 ggagaattgcttgaaaccgg aaggcagagg ttgcagtgag ccaagatcgt actactgcac 27840 tccagcctgggcaacaagag agaaactccg tctaaaaaaa aaaaaaaaag ttctccggca 27900 ttttttgaaaaaggcaaact gcactcataa aattttacct ttggaacaga atctttatag 27960 ttacataatcaatggaaaga acagatttga tgacaatatt gagcttatga attaatcaaa 28020 tttgaagctgctctacaccc agaattatta ttattattat tattattatt attatttttt 28080 gagacgacgtcttactttgt ctcactttgt cgcccaggct ggaatgcagt ggcgcgatct 28140 tggctcactgcaacctccgc ctcccagatt caagcgattc tcctgcctca gccttccgag 28200 tagctgggattacaggcacc tgccagcgtg ctcggctaag ttttgtattt ttagtagaga 28260 cgagctttcttttttttaag acggagtctc gctctgtcgc ccaggctgca gtacagtggc 28320 gtgatctcggctcactgcaa actctgcttc ccgggttcac gccattctcc tgtctcagcc 28380 tcccgagtagctgggactac aggcgcccgc caccatgccc ggctaatttt ttgtattttt 28440 attagagacggcgttttgcc gtgttagcca ggatggtctc gatctcctga ccttgtgatc 28500 cgcccgcctcagcctcccaa agtgctggga ttacaggcgt gagccaccgc gcctggccag 28560 agacgagctttcaccatgtt agtcaagctg tcctcgaact cctggcctca agccatccac 28620 ccacctcggcctctcaaagt gctgggatta caggtgtgag ctaccatgcc cagtttatac 28680 ccagtcttgttaagtgagat gttacatctc cctctgttta gttcacttga cgcaagattc 28740 tctatttttttttttgagat ggagtttcac tcttgttgcc cagggttgta gtggcacaat 28800 cttggctcattgcaacctct gcctcccagg ttcgagcaat tctcctgcct cagcctccag 28860 agtagctgggattacaggcg cctgccacca atacaatact tttttgtatt tttagtagag 28920 atagggtttcactatgttgg ccaggctggt ctcaaactcc tgatctcagg tgatccaccc 28980 acctcggcctcccaaagtgt tgagattata ggcataagcc actgcacccg gcctaagatt 29040 ctctattacttgagaataaa acaacctgtt aaaatattat accacagtgt gcttggccta 29100 tgtaacatctgcttagataa catactctct taagcagtaa atgagtatga gttacagggg 29160 ctctccttttgttctttagg gactctagaa atgccagata attccacttt tgtggtgaca 29220 gaagaatctggcaataatag ctaccgttta ctgaacaaca actgcacatt aagcactgtg 29280 tcatatgctttaggtatgtt atttgatcct caccaaatgc ctaggtatta ttcctctttt 29340 cttttcttttcattttcttt tctttctttt ctcttttctt ttttattttc tttcttttta 29400 acaaagaaagaaactgaggg ggctgggtgt ggtggctcag gtgtgtagtc ccagcatttt 29460 gggaagctgaggttggagga tcacttaagg tcaagaattt gaggttacaa tgagctatgc 29520 tagcaccactgcactccagc ctgggtgaca ggtgagactc tgtctctaaa aaataaataa 29580 atttacatctgttcaaaaga taaatgacct tttaaacaaa caacatgtag tataaagttt 29640 atgacatacaatcataaaaa ataattaata aaaaaaacag ccaatgtgac ctgatattta 29700 tagaacactcttaacaatag cagaatacac atttttaaaa gtacctgtag aacatttatc 29760 aaaataggccatactatttt tctcaataaa tttaaaatta tttctgtcat aaaatatact 29820 ttctggccacaatataatta aattagaaat caataaaaag gatatctaga aaatctccaa 29880 atgtttggaaaataaaactt ctatatcaca cattagtttc aaaaaaagaa attggaaagt 29940 gttttgaactgtctgaaaat taaaacacaa gataataaaa cttgtgagat acaataaaat 30000 agtgctagagggagtcttgt agcactaaat gcctatatta gaaaataggg gcccggcgcg 30060 gtgtctcatgcctataatcc tagcactttg ggaggccgag gcaggtgatg gcttgagctc 30120 aggagttcaagaccaacctg ggcaacatgg tgagaccgcc tctctacaaa aaatacaaaa 30180 attagctgggcagggtgtca tgcacttgtg gtctccgctc ctcaggaggc tgaggtggga 30240 gggtggcttgagcctgggag gttgaggctg cactgaggca tgttcatgcc actgcactcc 30300 agtctgtgtgacaaagcaag accccgtctc aacaacaaca acaaaaacaa caaacaaaca 30360 aacaaaaaacgaaattagaa aaagagtaag ttaaacacag aataaaatga agacaggaaa 30420 taattaagattggagcagaa acttatgaaa tagaaaacaa aaatagcagg aaatcaataa 30480 agcctaaagctggttctttg agaagatcaa taaaattaat aaatccctag gccgggcatg 30540 gtggctcacgcctgtaatcc cagcattttt ggaggccgag gcgggtggat cacgaggtca 30600 gaaggtgagaccaacctggc taacacagtg taacccagtc tctaccaaaa atacaaaaaa 30660 attagccgggcgtggtggtg ggcgcctgta gtcccaccta ctcaggaggc tgaggcagga 30720 gaatggcgtgaacccaggag gcggagattg cagtgagctg agatcgtgcc actgcactcc 30780 agcctgggcgacagaatgag actctgtctc aaaaataaac aaaacaaaac aaaacaaaaa 30840 acaggttaaaagaccggtgt ggtggctcat gcctgtaatt ccagcacttt ggaaggctga 30900 ggtgggcggatcacgaggtc aggagttcga gaccaccctg accaacatag tgaaacccca 30960 tctctactaaaaatacaaaa aaaattagct gggcatggtg gcacatgcct gtaatcccag 31020 gtactcaggaggctgaggca ggaggatcac ttgaacccag gaggcagagg ttgcagtgag 31080 ccgagatcgtgccactgcac tccagcctgg gtgacagagc aagactctgt cttaaaataa 31140 ataaataaataaataaataa attaaattaa attaataaac ctctagccag actgaacaga 31200 aaaaaagtgaaaggaaacac aaattgcaaa tatcaggaat gaaggagata acctacagat 31260 tctacagctattaaaataat aattagagaa tattatgaaa aactttttaa caaaaaattc 31320 aacatatataaaatggacaa accccttgaa aaaaaccaaa ttaccaaaaa ttgtacaaga 31380 agagctgacctgagtagtcc tatatctatt ttttaaaatt gaatttgtag tttaaaacct 31440 tcctacaaggaaaactccaa gcccagatgg cttcagtggt gaattatacc aaatgattaa 31500 ggagaaataacagcagttct ctaccacctc tttcagaaaa tggaagccaa tggaatactt 31560 cccaattcatcctaggataa cagcattacc ctgataccaa aacctgacaa agacattctt 31620 agaaaactacagatcagtag tcttcaggaa cacaggtgca aaaattctca aggaaatttt 31680 agcaaatctaacctaacaat atgtaaaaag gacaatgcat taagaccaac cggagtttat 31740 ttcaggcatataagtcttca tttcaaagcc caatcaatat aattcactac attaacataa 31800 aattaaaccatatgattacc ccaacagatc caccaaaagt gtttgacaaa atctaacatc 31860 cgttcctaataaaaactcag caaactaggt ataggggccc tttgtttgtc tttttctggt 31920 ttccaagtccttgaaacaaa atccaactat gtccaaatgc catgaaggtt tgtgttgctg 31980 ctgatgtcagagataaacat tacttttaag gacaggacgg agtggagtag cagaagcatt 32040 tagatgagaaaaaagacaaa ttaacttgtt taattcttct taagagccaa aatgcaggtg 32100 tttcttgcacaatgtagtat tctttttctt tttactttct tttttttttt cttttttttt 32160 gagatggagtttcgctcttg tcacccaggc tggagtgcaa tggcggatct cagctcactt 32220 ctgcctcccgggttcaagtg attctcctac ctcagcctcc cgagtagctg gtattacagg 32280 catgcgccaccatgcccagc taatttttgt atttttacta gggacggggt ttcaccatgt 32340 tggtcaggatgatctcaatc tcttgacctt gtgatccgcc tgcctcggcc tcccaaagtg 32400 ctgggattacaggcgtgagc caccgtgccc ggcctatttt tctgtagtcc cattttcttg 32460 cttcagagttattcaggagt tagcacggta ctacaattgc tatgcacaga agctgaggaa 32520 catttggtagtgttaaatac ctaacattga cttaaatctg tacataggta gttctagata 32580 tactatgcttctttactgca tcaaccagat ggacattaaa tggtagaatt atgactaatt 32640 tgtataaagcattttatata gtatatatat tttatttatt tatttattta ttgagacaga 32700 gtctcgctgtgttgcccagg ctggagtgca gtggtgcgat cttggctcac tgcaagctcc 32760 gctgccctggttcacaccat tctcctgcct cagcctccca agtagctgag actacaggtg 32820 tccgctaccacgcccgccta atttttttgt atttttagta gagatggggt ttcactgtgt 32880 tagccaggatggtctcgatc tcctgacctc gtgatccgcc cgccttggcc tcccaaagtg 32940 ctgggattacaggcgtgagc cactgcgccc ggcctagtat aataattttt aaaattagct 33000 ttaaatatttttgagttaaa atcttgatat tttaaaatgt tgcctattaa ttaatttttt 33060 ttttttttttttgagacgga gtctcgctct gtcgcccagg ctggagtgca gtggcacgat 33120 ctcggctcagtgcaagctct gcctcctggg ttcacgccat tctcctgcct cagcctccag 33180 agtagctgggactacaggcg cccgccacca cgcccggcta atttttttgt atttttagta 33240 gagacggggtttcaccgtgt tagccaggtt ggtctcgatc tcctgacctt gtgatccacc 33300 cacctcagcctcccaaagag ctgggattac aggcgtgagc caccacgcct ggccgcctat 33360 taatttttataagcagtttg cttttaatat tttagaagaa aatagctctt tgaatacatt 33420 taaaaccagttttaactttt taaattttaa tactttattt atttatttat tgtttgtttg 33480 tttgtttgacagaatgtctc gctctgttgc ccaggctaga gtgcagtgga acaatcacag 33540 ctcactgcagcctcaaactc ctgggctcaa gccatcctcc cacctcagcc tcccaagtag 33600 ctaggactagaggcatgagt caccacaccc agctaatttt taaaagattt tttttttgca 33660 gagacatggtctcactatgt tgcccaggct gatctcaaac tcctgacttc aagtgatcct 33720 cctgcttcagcctcccaaag cgttggaggt tacaggcatc agctactatg cgcaggtttt 33780 aatttacttttgaataagta tgtgaaatta aataattcaa acttaaagct gttggaactt 33840 tattctgagccttgagaggt gtgtggctgt gcagcctgag tcacatggca tgcagctgca 33900 acttttgccttgtttttcct ttagataatt aagaacaaac agcaccaaag acccccacag 33960 atcattacccctccttatag agtaataaag tattctttct tggaatttag caatctgtaa 34020 ccaatcaaattgctgtggca tatgcactag tcttgtatga aaagagtctt gctctgtcgc 34080 ccaggctgcagggcagtggc agtcatagct cactgcagcc tcgaacctgc cgggctcagg 34140 tgatcctcccacctcagccc tctgagtagc taggactaca ggcatgcacc actgtgccca 34200 gctaaatgtattttttgtag agatggagtt ttgccatgtt gctcagcctg tttttgaact 34260 gggctcaagcaatcctccca tctcagcctt ccaaagtgct gggattacag gcgtgagcca 34320 ccatgcccggccaaaaccaa ctaatattaa cagtattttg tgtgtctctc taaatatatc 34380 ctatgtgaatgtatgtatgt attctttctt ttgcctttat aaacaaatga tagtatattt 34440 ttcataacgttctgcactct gattttcttc tcaatgtatc ttggcagtct ttctcagtat 34500 atagtgacttttctcatttt tttatcttta tacctcaata tctggcacat agtaagcaaa 34560 tcataaatgctgagtgaatg aaatattaaa tgaataaaaa ggaaattttt gtgctgctat 34620 tggaaattagctctctatat atttcaacat gttacacata tacaatgatc taaaaacttg 34680 tcttactctttcctatccac tagagggaga catcaacctg ttgtggaaaa gaatgatcac 34740 ttaaagtctttagaaattct gaaccaactc tctagcaggt gatccttgtt agaatttgag 34800 cccttaacgctatccaggac tggaggttga agggacgata gagggagcag gaggagaatg 34860 cacatggattaaggagcgag aacacaggtg aacttcagct tttttgctaa cagtcagaca 34920 aactactgaccctgactcag tgatgtgcta gtaaaccagc tctttaaaaa aaaaaaaaaa 34980 gccctagattgctgatttgt atgtaatgtt tatgaatttc agtagagaaa aagacaatat 35040 tcaaactgagccatgcaccc aaaacaagag aacagccaag aagtgttcac ttctatcagt 35100 gccctgggttgtttgaaaaa agaagccgac ctgagcacct gtgagctccc ttctggcgag 35160 gagaaatctggagtgtagtt attccaccat ggccaaattc aagccactcg gggtttaatc 35220 accgaattgcaaattccttg aacatttaac agtaggctct cttggctggg cgcggtggct 35280 catgcctgtaatcccagcaa tttgggaggc catggcagga ggattacctg aggtcgggag 35340 ttggagaccagcctggccaa catagtgaaa ccccatttct actaaaaata caaaaaatta 35400 gctgggcgtggtggcaggtg cctgtggtcc cagctactcg ggaggctgag gcaggagaat 35460 cgcttgaacccaggaggcgg aggttgcagt gagccgagat tgtgccactg cactccagcc 35520 tgggcgacaacagtgaaact ccatctcaaa aaaacaaaac aacaataaca acaacagtag 35580 gctctcttgagccagcctga gcaggctctt gcatgctgct gaagcttgtc gggtcttagt 35640 tacttttcctgtaaagtggg gatgataaat ctgctcatta tgtagattct attacataga 35700 ggacacataagttctttgaa tgcttaaagc aatgtttcct aaacttcttt ggtcatgaaa 35760 tcacccagtggcttgtgtaa aataaacatt cccaggacct gccctagagc acctgggtta 35820 gaacattttgggggaggggg ctgggaatct gtattttaaa taagcaaccc aggtgaggcc 35880 gggcgcggtgcctcacacct gtaatcccag cactttggga ggccgaggcg ggtggatcac 35940 gagatcaagagattgagacc atcctggcta acacggtgaa attccatctc tactaaaaat 36000 acaaaaaaattagccgggca tggtggcagg agcctgtagt cccagctatt tgggaggccg 36060 aggcaggagaatggcatgaa cccgggagac agagcttgca gtgagccgag attgcgccac 36120 tgcactccagcctgggcgac agagcgagac tgtctcaaaa aataaataaa taaataaata 36180 aataaataaataaataaata aaaataaaaa ataaaaaagt gacccaggtg actcttatga 36240 ccctgtgaaatgggagaaac actgctgcaa attactctta taattgggtc aggtgtcagg 36300 ggtctttctctaacttcaca attgggcctg cttgaagaga tgtgtgcaga gttccacaac 36360 acactccaggcaggcattta atccgttcac tgtcttctct accctcagag cccaaacttc 36420 ccaaagaggaaaacctgctc cttgccatct cttaggccaa ggcttctgta cacctgggaa 36480 gtccttcaatctgaggatct ctgggttgtt ttcaagctac tatttattga gaatttacaa 36540 agtgtcaggcacgttacagc aatttgtcat ttctatgaaa tagcttcttg tgctattccc 36600 attttacagagaaaaatcaa agaagttggg aaaatgtcga agggcacaca actaggaagt 36660 gtttgtgctgaaaacccacc ctaggcccaa gccttggaac tccaagcctg ggttccatcc 36720 ctgcactgggcaattctgat ctatgtgcgc tagtttcctt gtgttctctg ttctctccgt 36780 agaaatcctgggctctcttc tcccagccac aaggttaggt tgaaaaacag agcagatgga 36840 ggtagtttgtagcctacagg tgccctgaat gaagcttcca cagtgctaaa gtggaagaac 36900 gagggactccaagggaagga ttcaaggctg ggcccatgca cctgtgtaat tcagaagaga 36960 ccccagaggagatcagcgcc ctctaattag ccctggtaag gagctctggg agttactgta 37020 actctctcagaagaacccaa acatgcggga acgtgacttc ttaccttctg aaagtccaca 37080 aaattcctgattgccaccat taatttgtca cttatcattt gcaacaggca ttgtaggttg 37140 tcttatgcatttgtcttctc ccttcagcta gtgtataaag tcttagggag accagcagtt 37200 cagagagaatgggctttggt gtgaaacaga tctggtttga accctctgct acttactagc 37260 tgttgggcaagttccttaaa ttctctgagt cttaatcttc tcatctgtaa aatggagaca 37320 taaggagtacccacctcatt ggattgtttt aaggataaaa ttaaatagtg caggcaaagg 37380 atttacaagcaactgctgaa tgaatggtag ttatagcctc ctcctcatca tctgtgagca 37440 aacaccctcatatttccttg tgtctcaggt agacacttaa ggtattgcaa gcattaaggg 37500 agcattgtcacaaagagata aatgcatgag ggcaagatgc agtctcaaag aagagtgttt 37560 tatgaaagaataaatgtaat gctgagtgtc agaaaaaaat tttttttttt aaagatgagg 37620 tatctatcacccaggctgaa gtgcagtggt gtgatcttag ctcactgaag cctcaacctc 37680 ccaggctcaagtgatcctcc agcctcagcc tcccgagtag ctgggactac aggtgccacc 37740 acacctggttaattgttgta ttttttgtag agatgggttt tcgccatgtt gtccaggctg 37800 gtcttgaactcctgggctcg agcgatcctc tcatcttggc ctcccaaagt gctgggattg 37860 caggcatgagccaccacacc cagcctgtca gaaaaatttt aaggtgaaaa taactaaaga 37920 agttgttaagaattttctcc cttgagtggt attttagact gagatgaggg agggtagagg 37980 taggatgagaaggaagggat ggggtccggt tgaaaggcct gtgagatagt agcagtgcaa 38040 tatggcagatgttgacagcc tcagtgctag gaacacagaa actgaatctc ttgcaaggag 38100 gcaggtgtgcatctgtatgg aagtcagatg acctgtgttc ctatgagtgc aaatctggaa 38160 aacaccctcaagtttccttg tcagcaaatt ggtgataaaa tcaacattgt agggttggtg 38220 tgaacatgcagcatgatgtg gccatgcaag ttctttgtta actagaagcc agtgtcatgc 38280 caggacagcagtcctcctag taagctgtgg ctggtggcgt ggtagaatac gtggagcagg 38340 ctgaggaagcacttgacttg actatgagca gaaccattaa gaagctagtt agctaaactg 38400 cctggacagtagaaaaataa tatgtgagaa tgtaaaagga agagaaacaa tgtgagggga 38460 gaggagaatgcagagatcct ggcccatgga acagcattgg tgatccttaa gtagctgcat 38520 gaactacttggagaagttca ttttctgttt ataattccca gcaaaggaga ggactgaata 38580 agagagaagaaaacgattcc tttctctggt taggttcatc agatcaaacg gtgacatatg 38640 tgaaagaagcacgctctgtg cacaaaaaat caagtctgta tttttataaa agccatttct 38700 gggctgggcgcggtggctga cgcctgtaat cccagcactt tgggaggcgg aggcgggtgg 38760 atcaggaggccaggagatcg agaccatcct ggctaccacg gtgaaaccct gtctctacta 38820 aaaaaatacaaaaaaattag ccgggtgtgg tggtgggtgc ctgtagtctc agctacttgg 38880 ggggctgaggcggaagattg tgccactgca ctctagcctg ggcgacagag cgagactccg 38940 tctcaaaaaaaaaaaaaaaa aaagctattt ctgtaatgag catcactgga gagttagttg 39000 ctatgggtctaaaggacaat atgaggcagt tatagtaact ttccatgata tgaacaaaga 39060 aattgaaaatgttagataca tttacaagaa gatgtagaaa aaactttagt caaaattttt 39120 gaaatattttttgaaatatt aaactatgaa atcagacagt cttatctatg gtctcaagcc 39180 atgtctgtctgtaccttttt tttttttatc tcatttcagg gaatattaca ctggctgact 39240 tattaatatcttctgagcca gaaaatgtaa ggaagctgca ttttcagaat tgcatttgag 39300 tcatttgtgaaattgcatat tacaatttgc cgccatttct aacagtccta taactttttt 39360 ttttttttttcttaactggg tgttcacatt catgccaatg acctctaggg gctagtttct 39420 cttctagctcaagagaattg ctgcagagtt ggaagtaagg acaaaaatgt gtatgcttca 39480 tgtttgatttcaaatgcata gaaaattaga aacttaaggt atgcaaggga tttgtgtgga 39540 atttaagtacctttgagggg cagtggacag gacaaaaagt tattttttac ctgtttgttt 39600 acaaatagcaaagatcaaga ctgaaacaca tgagtgtgat ttagaaagag ttggctgcag 39660 gtgctgcttgctcaggtggt tcatttaaac tgcaggtcag agcaaccttg tctcatggtc 39720 ctggtgcccaggtatcaggt tgggtctgtc ttgctgctta tgtccttgtt accctctgag 39780 ggccccagtccaacgcagat caataaagaa taagttacat aaatatgctc ataggtggtc 39840 attcctagacaagaaattga caacatttca ttcaacagta tctgggctct acaggacaga 39900 catgcctccatttatgcaac aaataagaac agcatctcat gacagtggag aaaacatggg 39960 atgtgcaggtaggtaggtaa agttgggtgg aaactttcac cctaccaaat gcacatgggt 40020 gactttataaaataaatgtt agctctctga gcctcagttt tcccatctgt aaaatagaca 40080 gtcccagggaattttcaagg attaaatgaa ataaaagtga atcaacctat gcaagcctgc 40140 ctactgtggtgtccaggcta gaaaaatgct caataaatat taggtttgtt tttatttcta 40200 caaaagatgtgatcctaaag agctctatcc aaattcaagt ttcaaatgtc aaatcacatt 40260 ttgtgaactttatgttcagt tgagatgatc tctgacatat taattagtaa tcctatcttt 40320 ttcattcatcaccaccaaaa aaaggtgtta ttgcacgttc aattaatctt tcccctttat 40380 taattccataagtgtagggt tttatctctc agattctctt aaaacagacc aatttatacc 40440 cacataatataaataagctt gttcctataa cactctggag cagataacta tcccagaacc 40500 caaatcctcctacttggctt caagctcaga gaataaagca acaatccaaa ggcacccttt 40560 ggcatgacacccttctagac atctgtagca ttcctccttt ccctccactt ttcctattag 40620 cttttgctttcttgcctttt acagggtttt gttttgcctc ttggtagttt ctttcctacg 40680 gaaaattctccctctgatct ttccaagtca aaggcttcag caaacatttg ttgaacgcgt 40740 ggattgtgctaggtgggtgt tatggaccat ggagaatgct agagatgtaa gacatgcgct 40800 gtccaatcgcagcgcaggtt gtgttgacag gtaagatgag ggctgtgggg gagccaatgt 40860 gcacgttccactgggctaat gtgctcttca ccttatttag gctcttggct ttgggatgtg 40920 taagactttgctagacagag aaggggtggg gtgagaagat gaggaaggtg caccttttat 40980 ggagaggctttccttcctct tcacagcaaa ccatacctgt actacattga cttcctttgc 41040 tttcccaggtgacatctagc tcatgctgca agctcatctt gttaatcata aatgctagta 41100 agttaatattacccatcata tataacatga cttaatttta acaattcaat gctttatccc 41160 caaaagatgacttaatggtg acaatttcaa tccccattgt aggatatttt ggagacaggc 41220 agtcctttcaatgtcatatg tgggtgcttc cttaggcagg tcaggggtga ggtggaaatg 41280 aggctgggaccctgctcact tatatagcag gcatcgttct caataccagg cttcaggggg 41340 ctttttggtctagccattgg tatgaactgc ctcaagaata atcccttcat cattgtggtc 41400 acaattcaggtagaattgga ataatcatcc tctccactct gcattaaacc aggcaaagtt 41460 tccatctctgggtaccattg tctttcttga tggacagggt gagtcagaag gaaacttact 41520 cactcccattcattttctgc ttattatttc ctgcagtgag gtttccttgt ataataaaca 41580 gcttctgtgggtgtttgagc tgctctgaaa agagaacatg ctgttcctgt gtgtagaatg 41640 ccttctgaaggaagcatcac agtgaacaca gagcagaagc ttggcacaca ggtggcagaa 41700 gtttgtctgcagtgttctgc atagagcaga gagtcaagcc attttcattc tgattgattg 41760 gaggcatggtatggaggtaa atgggtcctt ggcctctctc ctggattcaa gtccttctta 41820 gccactgataggtcatgtga ccatagggag gttgtttaac cttcctgaac attcattttc 41880 tcaagtataaaatgggggta atagaatttg ccttataggc ttgcgtataa aataagaatt 41940 attgagagaaagcggggcat aaatgtccaa taagcggtag ctgtctatga agccactgtt 42000 gttactgggttcctttctca ctaggtggct tcaggtagct gacagaagct ctgtgagcct 42060 caatttcctcactggaaaag tggagtcaat atctcactga gctggtgtga ggattaaatg 42120 agatgctgtgcaggtgctta gcacagcgtc aggtatgatg ttaatattga tagatgcatt 42180 ttcttcaccctcacctatct ttttctgcct gttggcttat ggttgaaatt ccttcatgac 42240 ggtttccatttccagagata tcttgttaac aagtatatac caccaaatga agctgatttt 42300 tttttttttttttttttttg agacagagtc tcgctctgtc gcccaggctg gaatgcagtg 42360 gcgcgatcttggctcactgc aacctccgcc tcccatgttc aagcgattct cctgcctcag 42420 cctcctgagtagctgggatt actggcatgt gccaccacgt ccagccaatt tttgtatttt 42480 tagtagagacgaggtttcac catgttggtc aggctggtct caaactcctg acctcgtgat 42540 ccacctgcctcggcctccca aagtgctgag attataggtg tgagccacca tgcctggcca 42600 tgaagctgatttttttaaac catcatttaa cattttctcc ataaggtggc aaggaggaag 42660 agcatatggggactgggtac tttgagagac cccaggacag gagacaggga ggctgagatt 42720 ggcatgttgtctgctgcagt tatttgccag cgacacactc ttcccgtcca aactaacttc 42780 tctgcctcaaggacagggag actctgcctt tcaacctgag agaaaccagg actctcagct 42840 ttaatgaaaattggacttag ggtggggcag tggagacttt tcacagctat tgtttagctg 42900 atgaagcagatgcttctcca tctttggagc ctgtcttcat tacctgtgga cctcatcttt 42960 atcaacccagagcacacttg cgtctctcta ttttggctaa acaccaaaca gctgaggctg 43020 gtactgtaaaactttccctc caaatgcccc ccctcgtctt cctctattag agatctggat 43080 cacaaccctcaaaaaccatg tcccttatgc cacctgagta gatggtttga tgattaatta 43140 ggcacagatgtgacactggg gggttctcac aatggcctgt gggtcacatg ctactttcct 43200 tttcattttcatcagcaaca gctgccttaa agccagttaa gactgtggtc ctagtctcgc 43260 accctggggctcctgctggg gtgggtgagg ggaacacccc attaagctgg gggaactggg 43320 gctgccaccagggggcgcga ggggccttcg cccgagaaga ggggtgggca ggtgcctcca 43380 gcggagaagggcgccgtggc cggaggcaca ggtctccccg gtgccacttc aagtgagttc 43440 gaggaagtacctgggatctt tgatctaacg cgaaaggcct tcccagtgac ctcttgagag 43500 ctgagaacccactccctcca cctctagtcc acggctttgc cactccaggg cccgaggtta 43560 cgtttgctgctggggatttg acaaacccaa agcctctctg gtttcaccac tggctcctta 43620 gaatcagacatctgttctga atgacactta tgtgagtcag gggctgagga cgtgatcctc 43680 gaagtgtggtccccagactg gctgtatcag tgtcggcatc ccccaggacc tggttggaaa 43740 tgcatattctcaggccctac tccagacctc ttaaatctga gactggggct gcggggagcg 43800 ccatctgtgcgccactatcc ttgtgggtgg accaggagtc ggttcgaggg tgctcccact 43860 tagaggtcacgcgcggcgtc gggcgttcct gagaccgtcg ggctccctgg ctcggtcacg 43920 tgggctcaggcactactccc ctctaccctc ctctcggtct ttaaaaggaa gaaggggctt 43980 atcgttaagtcgcttgtgat cttttcagtt tctccagctg ctggcttttt ggacacccac 44040 tcccccgccaggaggcagtt gcaagcgcgg aggctgcgag aaataactgc ctcttgaaac 44100 ttgcagggcgaagagcaggc ggcgagcgct gggccgggga gggaccaccc gagctgcgac 44160 gggctctggggctgcggggc agggctggcg cccggagcct gagctgcagg aggtgcgctc 44220 gctttcctcaacaggtggcg gcggggcgcg cgccgggaga ccccccctaa tgcgggaaaa 44280 gcacgtgtccgcattttaga gaaggcaagg ccggtgtgtt tatctgcaag gtaagcgccc 44340 cttcgctcgaggtgtggttt aattgtctca ttttgtttga aatcctgcgg tgagaaacca 44400 gtcgtgttgagaacaataaa agaccaaaaa acgatcacca aaaccaactg tcctgaaagc 44460 tactggaaagttggaaaatg catgctttga ttaaatgtct tcattcaaga cactggcaag 44520 ttaacttatttagtttgtgc cgtgagctct gggttgattg tgctaatatg aataactgaa 44580 aaacattttatttccctatg gttttcctcg atggacttcc ccactatggg tgaaatgaca 44640 atggagttgaatacactttc tgattgaact ttgagggcct gggaagatgt acacgtctca 44700 ggcaagatgataggggtttt aaaatgtatt aattggcatt ccttagccat gtcagcaagc 44760 tgcgttcctcctttcctggg cagaccaagc taagctctaa ctggtctcct ttatttgctg 44820 aagaggagtccaacaactgc cctctaacac cctgcgtgtt attcttattg gaaggacaat 44880 attaagtcaagtgaatgtca tttttgtgaa aaaactttga gtggacttct atttaggaag 44940 ataaggttgatttaatttta ctcgctgttt aaaaagcagg attgtgtttt ggtgtggtag 45000 gcaacattttggaggacaga ctttgcctta ttttgttata tttctagtat ttacatgggc 45060 attccattagaaagttttac ttttgctcta agtttcgtaa ctcggtgtct agtgagggga 45120 aacatgtttgtaatttaaaa agtgaacatg tgaaaggaaa ggcttttctg agagtgttgt 45180 aaaacaaatgtaacgtgact atgaaaagaa catgattaac atctttgact cctatttttt 45240 ctgaagaaaatgtattttga tatgagttct agaagaagga aactataagg atctgttcat 45300 caacaggcattagagtatac accgtaggat tgcattttac gttcaagcat ttttttagat 45360 gaatttctgaaacattctta ttttaaaagc catcagatgc ttgttaacac ttaagtcttg 45420 ctcaagacatagaagtttct gaaatcaatt aacatgttta ggacacattt cgtagtgttc 45480 tgagggatgtgaataaatct aatcacagtt tacatttctt aatgtattta taattcagaa 45540 aaggtagaatttagtagtaa attcaactca taaccatata attaacattt aatagatatt 45600 gatatgttcacttttaagaa taagaaggaa attttctata agtgtatgtt gaacacataa 45660 taattcaaaattcatgtgat aattttaggt gatgctttga gtcgttttat agaatataaa 45720 tatggataaaatataaaata ctgaaggctg aactcaaagt gtttaatgat aagtttttga 45780 taatacatctagaaaccttg agaattgtat gcttgaacgt tagatttcat aattcagtgt 45840 ctagcacattgttttatatg caatagcact ttaaaaaaat taggctacag cagtataatt 45900 tacatacagtaaaatttagc ctctgtaaat gtacctctat gaattctgac agatgcacag 45960 tcatgtaaccagcaccgcac acatgacaca gaacagttcc attaccccaa aagtcccctt 46020 tgtacctctacctaccccac tgcccctgaa aatcactgat caaaactaca taatgattat 46080 gtggttttgctctttagtac gtttttactt agacatattt tcctttactt cttttgaaag 46140 aaaaacctgtttttcccttt ttataggatg agtcagtttg tgctattttt aattctagta 46200 ccttgggataaatcaaggca aagacaatgc tatttgcaaa tgggaaactt gagacttgga 46260 ctaagtgttaaattcatata gggctaatag atttagttct tagcagattt agattctatt 46320 gtggtttaagcctttggtta tggcatatat cattagttat cctgaattga aatacaaggc 46380 cattaaaagttatttatatc atattaatag aatgcatcat tcttttataa tctttgaatt 46440 ttaaaacttctttattaaaa aaaaaactac ttttcattat acctgagatt aagaaagcta 46500 cctgaaattgcatattatca aatagtgaga agcaaaacag ggattgaaaa tgacaaattg 46560 aagacatttaaaatgcagag tgattacaat tgctgaaggt aaaatattta tcttcatagg 46620 ggcttaggtctgtgtccaac ttatttgtag atgtcaggat ttttaaattt ctgtgctcat 46680 gtcttgaagtctagattttc ctgcagggtg gagatgtata accttttgta aactaatatt 46740 tttcactgtttaacacagta ttcaattcag tatacagtta ggagcctgtt attggtaggt 46800 actgctaacatatatatata taaaattgat gtctttttcc tttttccttt gttctatgaa 46860 aaacagcctgtattttaaat atgtaactta ccttgcatac ccagttacag tggtagtaac 46920 taggatatgcagagtggcaa gtttatgagg agctagcaaa ctggatagtt ggccttccta 46980 gctggaattatgacaggtct tgaaaatgaa gggcttttag tggagaatct ttgtgtgggt 47040 gtacttgagagagggcagga gagttagggt gacctagaaa gatagattgc tggacttgta 47100 tatgtttcctcaaagccaga ctgcagcatt ttgttagtaa attgttgtgt gttctactgt 47160 caaacccaggcctggaaggg gagttgagtg cattcagcct aacttctgga ttggctgtgt 47220 catcttgaatcccttcactc ggaattctct ctgaccctgt cccaaatgaa tatttgaatt 47280 tggtccagttcctacagagc atggtctgtg gctgttgttg gtgttaggga agagcagaaa 47340 cttgctgttgagagagaaga cacttgagaa gactgatgaa ctctctccca cccctgcctt 47400 cgaggcttggtcctcctacc ctattcaaac ccttgaaact ctttcctatc caactaaata 47460 agcgccaattggttactagg agaattagct tttcctcatt ttagaaggaa acagggtttc 47520 cttatgtacatgttcttaag aattacatgc aaatcagtta ttaatgatga gttctctggt 47580 gattttggagtgttttatct tcctaatatt aaattaattg agggccttaa tattttgttt 47640 tgaaagaatatatttaaaaa ggctgggtgt ggtggctcac gcctgtaatc tcagcacttt 47700 gggaggcctaggtggctgga tcacttgagg gcaggagttc aagaccagcc tggccaaata 47760 atgaaaccttgtctctgtta agaatacaaa aaattagctg gccatggtgg ctcaagcctg 47820 tagtcccagctactcaggag gctgaggcat gagaattgct tgaacctggg aggccgagtt 47880 tacagtgagccgcgatcatg ccactgcatt ccagcctggg caacaaagca aaactctatc 47940 tctaaataaataaataaata aataaataag aatacattta aagataataa ttggccaggt 48000 gtggtggttcatgcctgtga tcacagcact ttgggaggcc gaggtgggag gattgcttga 48060 ggcaaggagttcaagatcaa tctgggcaac acagtgagac cctatctcta caaaaattta 48120 aaaatcagctgggcatgatg gtgcatgcct ttagtcccag ctacttgggg ggctgagttt 48180 ggaggatcccttgagcccag gagatcaagg ctgcagtagg ccatgatctt gccactacac 48240 tctagcctgagttacagagc tagagtataa cccccacccc ccaaaaaagc taataattgt 48300 caaacagctacttatgcaca tcaaggatgc ttgttgctta agaaatcttt ttaaatcttt 48360 tccatgaaattccttctagt tgctgctttg tgagcgtgaa ttttttactt ctgcaggaca 48420 cacaaatgtggagcatttga actgaatgct tgggaaagtg tgatgggcag gtggaagaag 48480 aatagggatgaggacttatc ctctattctt atcctcctag acttatcctc ctagtctgca 48540 agcttgagaatatggcatca ggaatatgtg gcattttgtc cacacacaca gtgttggcag 48600 gctaccagcagcccagctat ctggactagg ggtgatggat ttctgtggac agaagtcaaa 48660 aagtaaaattaggaggcaaa aatcttcagg gtggccataa agacattgta acttgtctgg 48720 aaattccaaccaacactaaa tgtgtatcca gtgatatacc aatagactgg cttcatcttc 48780 ttggatgtgtaataatacct tacagaatgc tttctttttt tttttctttt tctttttctt 48840 tattttttttgaaatgaagt tttgctcttg ttgcccaggc tggagtgtaa tggcacaatc 48900 tcagctcactgcaacctcca cctcccaggt tcaagcgatt gtcctgcctc atcctcccga 48960 gtagctgggattacaggcat gtgccaccat gcccggctaa ttttgtattt ttagtagaga 49020 cggggtttctccatgttggt taggctggtc tcaaactccc gacctcaggt gatctgccca 49080 ccttggcctcccaaagtgct ggggttacag gcgtgagcca ctgcgcccgg cctcagaatc 49140 ctttcacagacatcatctca tttcaccctc agagcaccgt gaaaaggtac agcaccaaat 49200 aggtacctgattctactgaa gaagatgtgg cagctcaggg agtttgtgga tttgtctaag 49260 attgcctggctttcaggcag agctggggct agaatgaatg ttctgctcta tccattgata 49320 gaatatacataagaacaggc ttgatggtgg ctgacctttt tttttttttt ttttttgaga 49380 cagagttttgctcttgtcac ctaggttgga gtgcagtggc gtgatctcgg ctcaccgcaa 49440 cctccacctcctgggttcaa gcgattctcc tgcctcagcc ttctgagtag ctgggtttac 49500 aggcaagcgctgccacaccc ggctaatttt gtatttttag tagagactgg gtttctccat 49560 gttggccaggctggtcccga actcctgatt tcaggtgatc tgcccacctt ggcctctcaa 49620 agtgctgggattacaggcat gagccacccg cgcccgggtg actgatttct tattaactag 49680 atttacaggtgctttgataa aaaccagtct agtcttggct ggcacggtgg ctcatgcctg 49740 taatcccagcactttgggag cccaaggcgg gcgggtcacg aggtcaagag atcaagacca 49800 tcctggctaacatggtgaaa ccccgtctct actaaaaaat agaaaaaatt agctgggcat 49860 ggtggcgggcacctgtagtc ccagctactt gagaggctga ggcaggagaa tggctgaacc 49920 cgggaggtggagcttgcagt gagccaagat tgcaccactg cactccagcc tgggcaacag 49980 agcaagactccatctcaaaa aaaaaaaaaa aaaagtgtag tctttttgga gtgtttttct 50040 gccatttctagggccaaact ttttcttgtc catgaatcat tgtcaaaatt gggaatttta 50100 aatactacttttttctttta attcaaaagc catagtatgt ttcccagcca gtacattaga 50160 acaccatgcacgatcccatg tgtacaaaaa gctttctggc tgaattcaga tgtgacctga 50220 gagggccaaatacaggggtg tgtgctggga gagagagaga ggtctctgga cagaaaacaa 50280 agcctgttcaccacccagga tatggaccaa ctattttagg ttatggtgac taaagaaaat 50340 tgacatgcaaataaatgaat aattcttaga atcaggatgt ctgggtactg gttctttggt 50400 tggccaggtgaaattccatg ccaggcccaa caattaaact ctttagagac aattttttcc 50460 tgttgtaccagaacattgta ctgaggccat gtttgaacat tcaatcgatg tgttgggaaa 50520 actctgccctacaatgttaa agaaattaaa tcttttgggg agtctttcct ttgaccagtt 50580 tatatctctgttttagagga gggcttctca accagaatgg gtttgttgac ttatttttac 50640 agacctctggtagaaaggag gtcttttttt gctacctgtt ctcctgtctc agagaactat 50700 tacaatggtgtaagttcatc atttcttccc cttattatgg ctctgcttag gaagaaaaac 50760 tctttgcattggctaccaag tacctaacta ttcaagatgc cactgacaaa gagttaatct 50820 gtgaatcatgtgaatctgat atatctgaaa tatatccaaa caaaaagcac ctagcctttt 50880 aatgactctccagaagtcag ttctctaact ttaattatca tccttctggg gatatgtgga 50940 aattctacagaagttgattg gtgatatgtt gagatgtgag atctgtattt tctaagcaaa 51000 gttgccatgcacctgattga ttggctaggt gtatcctggc atttgtcatt tgttggtggg 51060 gtctgatagttggtttcacc actgctgggt acccagagtc atcacatcca tagagacaga 51120 atgtaggctggtggttgcca ggggctgggg gaagggagga gtggggaatt tgtttaacag 51180 agagttttagttttgcaaga tgaaatgagt tctagagatt ggttgcacaa taatgtgaat 51240 atccttaacactactgaact ttatacttag aaatggctaa gatggtaagt tttatgttac 51300 atgtattttaacacaattaa aaaagaaaaa aaaaaaaaca acttcaggcc aggcacggtg 51360 actcacacctgtaatcccag cactttggga ggctaaggcg ggcagatcac ttgaggtcag 51420 gagttcaagaccagcctggc caacatggtg aaaccccatc tctactaaaa atacaaaaat 51480 tagcctggcctaattgtgca tgcttataat cccagctaat tgtgaggctg aggcagggga 51540 atcgcctcaaaccctggagg tggaggttgc aatgagccga gatcacacca ctgcactctc 51600 cagcctgggtgacagagtga gatttcattt caaaacaaaa aaccacttta gaaactgcta 51660 gttttggcaatagttatcac tatatgtttt atcctgcata ttttctgtta agaataagga 51720 attgtttatgttgatcagga atctaagtaa ttaaaataca aaattctggc tggtggctct 51780 cgcttgtaatcccagcactt tgggaggcca aggcgggtgg atcatttgag gtcggaagtt 51840 caagaccaggctggtcaaca tggtgaaacc ccatctctac taaaagtaca aaaaattagc 51900 tgggcatggtggtaggcacc tgtaatccca gctactaggg aggctgaggc aggagaagca 51960 cttgaagtcaagaggcggag gttgcagtga gccaagattg taccactgca ctccagcctg 52020 ggtgacacagcgaaactcca tgtaaaaaaa aaatgaaata taaaattcca tactcattat 52080 taattacatatagtattaaa ataaaaccca aacaccaaac cttccttgat cctatatcct 52140 tctccagctaccattctctc tcctctcctt ggtccaaatt tttgatttac aatgttggtt 52200 ggaagtggtaccactttggt gttagttcct tatcatttta cctggtctgt cctgcctctt 52260 cctggtacattagctccctg aaggcagggt gtatgtccca gaactccttg aagtcccttt 52320 tctcagcatactaccatgcc tactgcagca ccccccatct ttaatgtcct tgacttggtg 52380 aaatattacattttgaacac atttcctcac ttccttatga caaatattga ttgagtttca 52440 gtgcaaggtgagtaagaaat ggtacttgct ttcaaggagc taaaaatctg aatttccttt 52500 tttttttctttttctttttc tttttttttt ttttttgaga cagagtctca ctctgtcacc 52560 tgggctggagtgcagtggca cgatctcagc ttaatgcagc ctccgcctcc cagattcagt 52620 gattctcatgtcttagcctc tcgagtagct gggactacag gcatgcacca ccacgcctgg 52680 ctaacttttgtatttttagt gaagatggtg tttcaccatc ttggccaggc tggcctcaaa 52740 ctcttgacctcatgtgatcc acccacctcg gcctcccaaa gtgctgagat tacaggcatt 52800 gactttacttcttactctcc tatgcacctc tatcattttg aagaagggtt caaggtagtt 52860 ctgataagcaggattaggtt tgtatgtaag tgattaaagg ggtgctatga gcaaaaaaag 52920 tgtgaaggtataacaagcca accacctcac aatgcagttt gcatgtttct taatggacat 52980 agcaggttttctgtaagaaa acagcaggag attcgtgtgg aatgatgggt tgaggcaaca 53040 tagtggcatcccttgaatgc tcgaagaatg tgacttagag tttggtggga agcagagagc 53100 tgggttttaagaacatgaat ctgacaactc tatggatctg gaggagaagc taactgggga 53160 cgaggagcagtaagaagcct gttacagatg cactgataag aagtaatgag agctggccgg 53220 gcacagtggctcacgcctgt aatcccagca ctttgggagg ccgaggcggg caaatcacaa 53280 ggtcaggatttcaagacgag cctggccaac atggtgaaac gccgtctcta ctaaaaatac 53340 aaaaagttagctgggcgtgg tggcgggcgc ctataatccc agctactcgg gatgctgagg 53400 cagaagaatcgcttgaacct ggaaggtgga ggttgcagtg agccgagatt gcgccactgc 53460 actccagcctgggtgacagt gcgagactcc gtctcaaaaa aaaaaaaaaa aagtaatgcg 53520 ataatgagagcttacttcaa gatggcagca aaagacagtg gaaaaaaggc attgggaaaa 53580 aaagccaatgtgccttgatg agtaaagtta actgagtcaa ggggagaagt caaaggtaac 53640 tatgatgggctttttctatt aacacaaata ggaaatgagt ggttttggga aagaaagtga 53700 tgaattacccctcagatatt gtattaattg tctattactg tggccgggca tggtagctca 53760 tgcctgtaatcccagcactt tgggaggccg aaacaggcag atcacttgag gtcaggagtt 53820 cgagaccagcctggccaaca cggtgaaacc ctgtctctac taaaaataca aaaattagtg 53880 tggtggtgtatgcctgtaat cccagctact caggaggctg agacatgata attgcttgaa 53940 cctgggaggcagagattgca gtgagctgat atggcgccat tgcactccag cctaggcaac 54000 aagagtgaaactccatctca aaaaaaaaga tttgcctgta atcagccagc acccccagcc 54060 ttgtgctcactttacataca aaaattctgt tttttagagc ataaattgaa gggcacattc 54120 aaaactgatacgtaggccag gcatggtgac ttatgcctgt aatcccagca ctttgggaga 54180 ccgaggcaggtggatcactc gagatcagga gtttgagacc agcctggcca acgtggtgaa 54240 accccatccctactaaaaaa tacaacaaat tagccagtca cagtggtgcg cacccatagt 54300 ctcagctactcgtgaggctg aggcaggaga atcactagaa cctgggaggc aggaggttgc 54360 agtgagccgagatcatgcca ctgcactcca gcctgggtga cagagtgaga ccttgtctca 54420 aaaacaaagacaaaaccaaa acaaaacaaa actgagaagc aacagattga taagtgacac 54480 agttacactggtcagtctct tcagctaata cccattgttt tttattattg gagattcata 54540 atgtgttttctttcttttaa aaactttttt cggaaatggt aatttctctc tttttttttt 54600 tttttttttttttttgagac agggtctcac tctatcaccc aggctggagc gcggtggcac 54660 aatctctgctcactacaacc tctgcctcct gggcttgagc aatcacacct cagcctcttg 54720 agtagctgggacaacaggca catgccacca ttcctggcta atttttagta gagacggggt 54780 ttcaccatgttgcccaggct ggtctcgaac tcctgacctc aagtaatctg cccacctcag 54840 cctcccaaagtattgggatt acaggcgtga gccactacgc ttggcctcat agcgtatttt 54900 aatattggttgagactagcc ttgctcattg atcttctctt agcgtttact tggttattct 54960 tgcttatttttccataagaa ctttcatttt tatttaatcc tgtgtttttt ggttttaaag 55020 actattttataataaatttt cgtgattaaa ctcttgtgct taaactcttg attaaacaaa 55080 caagcaatgaagagatgaat gaagcagaaa atgtgagttt catgcctcac attcccactc 55140 ctctgaggttaatattttca tgtatatttt tcaggatgta tttgtaatct catacaaacg 55200 tatgtatttttttaatgaaa atatttaaat tttcatagtt aacagctgta gctctaactt 55260 ggcaatatcttctgtgtttc tttacagcca ttatacttgc ccacgaatct ttgagaacat 55320 tataatgacctttgtgcctc ttcttgcaag gtgttttctc agctgttatc tcaagacatg 55380 gatataaaaaactcaccatc tagccttaat tctccttcct cctacaactg cagtcaatcc 55440 atcttacccctggagcacgg ctccatatac ataccttcct cctatgtaga cagccaccat 55500 gaatatccagccatgacatt ctatagccct gctgtgatga attacagcat tcccagcaat 55560 gtcactaacttggaaggtgg gcctggtcgg cagaccacaa gcccaaatgt gttgtggcca 55620 acacctgggcacctttctcc tttagtggtc catcgccagt tatcacatct gtatgcggaa 55680 cctcaaaagagtccctggtg tgaagcaaga tcgctagaac acaccttacc tgtaaacagg 55740 taagtccagtcttcattctg aattatagtt gctagccatt tctcaaatca ctttatggtt 55800 gagtgagaaggaaataatat gttagacaag gtctttattg tattaattac atagtttact 55860 tacagcacccaaaacacagg atgccctgtt ctattctgat attttagttc tcattaaaaa 55920 ctggtatgtgtacatcagtg ttgtggggag aatttgctat catgactatt gtctttatac 55980 agtaaatactgaacttaagt cactcctttt ctttttttga gacagggtct cgctctgtca 56040 ctcagactggagtataatgg cacgattgcg gctcactgca accttcacct cctgggttca 56100 agcaattctcgtgccttagt ctcccgagta gctgggatta caggcgcgtg ccaccacgcc 56160 cagctcattttttaaatttt tagtagagac agggtttcac catgttggct aggctggtct 56220 tgaactcctgacctcaaatg atccacctgc cttggcctcc caaagtgctg ggattacaga 56280 cgtgatgaacactgtgcctg gtctgaactt aagtcactct taatggagtt atttggattt 56340 gaaaaatgaatttttacttt actttcagtt tcaaagtctt cttatagtga aaccacaatt 56400 taatgttcatgacaaattgt ttccaggata aaagtaactg tgatagtatt acaacttaaa 56460 tgaaattctagacatgcgaa gcatgaaaag atagatgatt ggtataagct ttttaaccat 56520 gaactaaaataataacatta tataaagatt ggtggaaact attgaagttt aggcttcagt 56580 tgacattccctgaagttaaa aaggatatgt gtactcttta aatgcaaggt aacataatgg 56640 attatttccatctaattatt aatatttcta atgataatca taggtatgaa gggaatggat 56700 agtataatgagaaaggagag ggggagataa aaatctaaaa gtactaaggg catgttggat 56760 attgaaattcactactttca aatattatca taaaactttg agacagtaac attgcaccat 56820 tatttttcttcttttaaaaa cattttactc attggtaaag agaatataaa cattgtggat 56880 aacttttttaaagtaatggt ttgttttttt tttctccttc ctcctttaaa ggaagacata 56940 ttttgtttctgagcatgaat tataatcaaa gttctgctaa tttttgggca aattaatcca 57000 ttatataattaccttcattt ataaatcaat aataccttta ccattccctt tccaaaagaa 57060 ccatgcctggcaacatcagg aactagccag atgtgttttg gaggctgcct ggggatccct 57120 tgttagacttttcgttcctt tatgaacctc ttgcctgtgg tccagcattg agcctctgct 57180 tccttccaagcctttccagg ccaggcactt gcttgttctc tctcttctct tctctcttct 57240 tttttctctctccctttctc ttctcttccc cctttttctt gtctcacatt catctcaagg 57300 taacttaaagtccatttgtt attcctctta aagttatttt tattttattt ttttgagatg 57360 gagtctcactctgttgccca ggctggagtg cagtggcaca gtcttggctc actgcaacct 57420 ctgcctcccgggttcaagca atctcctgct tcatcctcca aagtagctgg gattacaggt 57480 gtgcaccaccatgtctggct aatttttgta tttttagtag agatagggtt tcaccttgtt 57540 ggccaagctggtctcgaact tctggcctca ggtgatacgc ccaccttggc tccccaatgt 57600 gctaggattacaggcatgaa ccattgcgcc caacctgaaa gttattttaa atctagacct 57660 ttatctgaaattgcagagtg tgagatgttt gttctccatt taaatgggaa cttcaaatgt 57720 ctgaagggctgcttagcaat gctgttggga atgactgatg tttggaagtg gttgaatgcc 57780 ttcacacccatccatgcagc attcgtgaac tctagtaact acagaagacc aatgcatatc 57840 ctgcctgtggttcagacctg tgggtaagat ttgatctggc cactcctttc attacactta 57900 gagatgtagctcccacccca tggctatgac tggtcttcgg cagtgacaaa tgctcatcag 57960 catcacgtggatgggcataa actcacctac ccactttcaa acattagtca ttccccacag 58020 cgtggctctttgtagatatg atatcagtat caaaagcttt gctgtatcag atttccggga 58080 atatatttaccaggaaccct ggaggaaaaa gagattaaat taggcaatgt tcatgctatt 58140 tttttttcctagaaagccct tcctttccct tttatgctct gttcaatgga tattttcttt 58200 gctccctagagagacactga aaaggaaggt tagtgggaac cgttgcgcca gccctgttac 58260 tggtccaggttcaaagaggg atgctcactt ctgcgctgtc tgcagcgatt acgcatcggg 58320 atatcactatggagtctggt cgtgtgaagg atgtaaggcc ttttttaaaa gaagcattca 58380 aggtacaagagaattgttaa ctgcttcttt agtttcctac ttttgatttc aaacaatttt 58440 gcagagatgacttggcagaa atgtcactac tggcctgttt ggcacacaaa gtatttgatg 58500 agcagttcagaggatcatgt gtgtttggaa gtgggttggg tggtggggtg gaattgcaga 58560 tttctaccccagaaccccaa gattatacag ccaactcgaa tgggtcttac ccctcgttca 58620 cccacatgggtgttggatag aagacatcga gttacaacct tgtgaagatg tctcttggaa 58680 aaaatgtgctcacaaggagt tgcaaagatt gtttctttct tttacttaaa tttaatatat 58740 agcatgcttaacagtcatga tggtgggctg gctcctgagg aagaaagaat aaacacattt 58800 tttggaaatggtcagaaatc aggaattcag ctacagtgga ctttgagaat tgatctagac 58860 acatttcttcccctaggcta ggagggtctc agttcacaat ccccttgttt tctgggctgt 58920 gtttagattatttccctaac tttctctaaa cgccttctgg attttttttt ttaaatcaac 58980 ttgttgatgaaaagaatcaa actctgtaaa atatttgaag agatttattc tgagccaaat 59040 atgagtgacaaatggcctgt gacatagccc tcaggagatc tgagaacatg tgcccaaggt 59100 ggtcaggccacaacttggtc ttatacattt tagggagaca taaggcatta atcaatgcat 59160 gtaagatgtacattgattca gcctgaaaag gcaggacacc tgaaagcagg ggcttccaag 59220 tcacaggcagttcaaagatt ttctgattgg caattgattg aaagaattat tatcagtagg 59280 aagcaatgattgggttacaa taagggattg tggagaccaa ggttttatca tgcagatgaa 59340 gcctccaggtagcaggcttc agagagaata gattgtaaat atttcttagg ggtcttaaag 59400 ggtctgttctatcagtgatt ccaaaagggg agggagggta taatgaacca tgtctgtctc 59460 ccttgttccatcatggccta aacttatttt tcaggttaac tttgtaatgc ccttggccaa 59520 gaggagggacccattcagat ggttgagggg ccttagaatt ttattttttg gtttataaac 59580 ttcaagttgtgcacccctga tttcaaggct ggtcagctca tctccctgca tgtgtctttg 59640 ctacactccttctctcgtac cagccctgat ttgctgaagt cactttcttg cttactcttg 59700 ttttctctatttgccccata acctgtccct caactgctcc ctcccaggca acaccctatg 59760 tttccatctgaaagctccct tcctttttct atcaaagccc caatgctttg ttctttgcca 59820 gttaagaaaagcaacgttga gagaattcat agtgtgtaaa tggcaaatag caatttacta 59880 aattaactcacccattgata actctaagag gatgttttac cttaagcaga gaaatactga 59940 tagaatccaggatatggtga ggagtgaaat gttggtagtc accttcctac ctgtcccctg 60000 aaattcaccctgtatgaatg gcagcctctt tgtcctggat tttataatta ctagctctgc 60060 gacttcacctcctagcctgt ttcctcctct gtgaaatgga gatactcata gggattttct 60120 aaagatgaaataaggttgat tatatgaaaa catattcagt gctcaaatat tttatttgtg 60180 acaatcttaacagtagatta taaggccaag tccatttcct ggctatatga taagaacaat 60240 attgattttctgaaattctg aactgaattc ttgatacgat gactattttg tatcttgctg 60300 agtttctaggattttacccc ttaagaacgt ttggacctat tactactaac catatctttt 60360 aaaaagagatccttcttttt tttttttgct tttggggaaa cattggtctg cttgaaacat 60420 ctttgacccctgagactaca gctaataaca attgaaagta aatttccttt gcttctctat 60480 gttgtttcttccttcctgct gcatcagaca ggaatgtcaa attctaaatg tgcaaagagg 60540 aaagagttaaagctgttaca gttgtacagt tgtagtgcct aaatgatcct ttctttgcat 60600 gcttcctgtctttgatataa gtgcattaca gtaactgaaa gtggccactt atttttaaaa 60660 ttgtctcaaataggccagga tggtacagta ttgagaaatt ccttgcatgt aacttttttt 60720 tttttttttttttttttgga gatggagtct cattctgtca cccaggctgg agtgcagtgg 60780 cacaatctcggctcactgca agctctgcct cccagattca caccattctc ctgcctcaga 60840 ctcccaagtagctgggatta caggagctgg ccaccacacc ctgctaattt tttgtatttt 60900 tagtagagacagggtttcgg catgttagcc aggatggtct cgatctcctg accttgtgat 60960 ccgcccgcctcggcctccca aagtgctggg attacaggtg tgagccaccg cacctggccc 61020 attcttatgttttttataat tttaaacttg tcttgctaac ttgatttata agctaattga 61080 ccatatcttagtcatgtacc tgtccccttc actgtacaaa tgcactggaa gctgtgttgt 61140 gcttgcttttccattgatac tttgttggct tcttcacaca atgagttgcc atcagagtga 61200 taagtgctgttgtttctcta ctgggttatg gagcacagag gaaggaggac atagggagaa 61260 ggacctcatcacttcatctg gtccagatga cagcatggct tattttttga gcttatcctt 61320 tactttttgttctctttcct attggtgttc atttaacaaa tatttattga gcattgtacc 61380 aggctctacggatgcagtgg tgaacaagac aatagattat agattccatg agggcaagga 61440 tttttgtccattttgttcac tagtggcact taccaattcc ttgaatatga tttttcaaaa 61500 ttaattgggcttatacacag agttctgtat catttttcac ttaatattgt cgtataagca 61560 tttctgttgttaaatttcca gagacccttt tcacaactgc acaactgtgt aatattctat 61620 cttatgatcagatttaattt atttaactct ttattattga aggccctatc aattattttc 61680 attgttttaatgctatggtt actttatttg ttcatgaagt tttgaaaaaa ataagtttct 61740 ctggatatgtttttagaaca aatctgtggg gtcagagtgc attaatgttt aaagttcttg 61800 acagatgttttcaagtgttc aagtcttaag aaggttgtac agacttgctc ttttaccagc 61860 agtgtgagtgtcgctttttc caacctcttg gtagcattga ctcttatcaa aaagaaaaaa 61920 accttgctacattgatacgt gatgtatagt atcttttggt ttcaatttgc ttctctttat 61980 tagtgaggtaaatgttttct cataaatcta tctgccattt gtattttctc ttttatcttc 62040 tttattcagagattttgccc gtttttatat tgggttctgg catttgcttg ataaatttat 62100 tgtgtgctttatatattaac ctattattac atgtatgaca aatatttttt ccacttgact 62160 ctgattttgatatgcagaaa tagttaatct ttaaatagtc aaatattacc aactttgata 62220 gttttgtgtatagtttttaa gcctaaaaaa agtcctttca tacccaggca ttatataaac 62280 ttttgcgcatattttgttat ttaatagttt gtttttacat tttattattt aattcaaaag 62340 aaatttattttggcatactg aatgaaataa ccaaatgtat ttatctctcg ttaattttct 62400 ccacatcattttacttaata aatccattca tttttcattg atttaaaata tgggagccaa 62460 tttttaaaagttgagtttga gatataatat gcatacaata aaatttacct attttcatta 62520 ctggtaatagtaagtacata gaaaaccaca gaataatata acactgttaa ttgtggtttg 62580 taacctcatattttgagtag aaagtctaaa ggaagaacca atgaaaaaca ataactacaa 62640 ctttttttttcttttttttt gagacagagt ctcactctgt catccaggct ggagtgtaat 62700 ggtgcaatctcggctcactg aaacctccga ctcccaggtt caagggattc tcctgcctca 62760 gcctcccaagtagctgggat tacagacacc caccaccacg cctggctaat ttttgtattt 62820 ttagtagagatggggtttca ccatattggc caggctggtc tcgaactcct gacctcagga 62880 gatcagcccacctcagacac atgtagattg aaaataaagg gatggaaaaa tatttcatgc 62940 aaatggaaaccaaaaaagag caggagtggc tatacttaga ccagaccaaa tagagttcaa 63000 gacaaaaactataaaaagag acaaaaaagg tcactaataa taaagatgtc aattcagcaa 63060 gagaatataacaattgtaaa tatgtctgga gcacacagat atataaagca aatattatta 63120 gagctaaagagagagacaga ctgatatggt aatagctgga cactttaaca ctccactttc 63180 agcatcgaacagatcatcca gacacaaaat caacaaagaa atgtcagatt taatctgcac 63240 taaagaccaaatggacctaa tagatattta cagaatattt catccagtgg ctgcaacata 63300 cacattcttttcctcagcac atggatcatt ctgaaggata taccatatat taggccacaa 63360 gacaagtgttaaaacattca aaaaaactgg aatcaaatca agcaccttct ttgaccacaa 63420 tggaataaaactagaaatca ataaagaatt ttggaaacta tacaaacatg gaaattaaac 63480 catatactcctgaacaacca gtgtgtcaat gaagaaatta agaaggaaat taaaaatttc 63540 ttgaaacaaatggtaatgga aacaacatac caaaacctat aggatacagt gaaagcagta 63600 ctaagaggaaagtttatagc ttaagtgcct acatctaaaa agtagaaaat cttgaagtaa 63660 acaacttaatgatgtatctt aaagaactag aaaagcaaga gcaagccaaa cccaaaatta 63720 atagaagaaaagaaatattc ataaaaagat caaagcagaa ataaatgaaa ttgaaaccaa 63780 gaaaacaacacaaaagattg acaaaatatg aaggtttttt tgagaaaata aacctgacag 63840 atctttagccagactaattt ttttaaaaaa aagatagaag agtcgaataa aatcagatga 63900 aaaaggagatgttacaactg ataccgcaga aatctaaagg atcattatag gctattataa 63960 gcaactatatgataataatt tggaaaacct agaagaaatg gataaattcc tagaccacat 64020 acatactgttaagattgaac tatgaagaaa tccgaaacct gaacatacca gtaacaagta 64080 acaagattgaagctgttata aaaagcctcc cagcaagctg ggcacaatgg ctcataccta 64140 taatcccagcactttgggaa gccaaggcag gaggatcacc ttaacccagg agttcaagat 64200 tagcctggacaacacacaga gatccctatc tctacaaaaa aaaaaaaaat tacaaattag 64260 ccaggtgtggtggtatgcat ctgtagtccc agctcttcag gaggctgagg tgggaggata 64320 gcttgggaccgggaagtcaa gactgtggta agacaagatt gcaccactgc attctagcct 64380 gggtgatggagtgtgatcag gtctcaaaaa aaaaaaaaaa aagtctctca gcaaagaaaa 64440 gccaggactgatggcttcat ccagaatttt accaaacatt taaagaagaa ctaatgccaa 64500 tcctattcaaacaattctga aaaatagaga aggaggaggg aataatttca aaatcattcc 64560 gtgagaccagtattaccgtg ataccagaac caaagaaaca tcaaaagaat atgacagacc 64620 aatatccccaatgaatattg atgtaaaaat cctcaataaa atacaaacca aatgcaacaa 64680 cacgttaaaaagattattca tcataaccag gtggaattta tcccagggat gcaaggatgg 64740 ttcaacatatgcaaattaat ttgatgcacc atatcgacag aatgaaggtg gaaaaccata 64800 taatttcaattgatgctgaa aaggcatttg ataaaattca acatcccttc atgataaaaa 64860 cccttaaaaaactgggtata gacagaatat acctcagccc aataacagac atataacaga 64920 cccacagctagtatcacact taatggagaa aaactgaaag cctttcctct atatggaaca 64980 tgacgaggatgcccactttc accactgtta ttcaacatag tactggaagt cctagctaga 65040 gcaatcagaaaagagaaata aagggcatct aaattggaaa ggaagaagtc taattatcct 65100 agtttgctgatgatcttata tttggaaaaa ttgaaaaatt ccaccaaaaa actattagat 65160 ctaataaattcagtaaagtt gcaggatcag tagcatttct atatgccaac agcaaacaat 65220 ctgaaaaaaaaatctaaagt gatctcattt acagtagcta caaataaaat acctgggaat 65280 taaccaaataagtgaaagtt ctctacaatg aaaactataa aacactggtg aaagaaattg 65340 aagaggacaaaaaaaatgga aagatattcc atgttcatgg aatggaggaa ttaatatgtc 65400 catactacccaaagcaatct acagattcag tgcaatttta tcaaaatacc aatgatattt 65460 tcacagaaatagaaaaaaca accctaaaat ttgtatggaa ccacaaaaga tccagaataa 65520 ccaaagctattctgagcaaa aatatcaaaa ctgtggaaga atcacattac ctgactataa 65580 attataccatagagctatag caaccaaaac aacgtggtac tagcctaaaa cagacatagg 65640 gatcaatggaacagaataga gaacccagaa acaaatccat acatctacag ttaactcatt 65700 tttgaaaatagtctcttcaa taaatggtgc tgggaaaact ggatatccat gtacagaaga 65760 ataaaactagatccctatct ctcaccatat acaaaaatca agtccagatg gatcagtgac 65820 ttaaatctaaggcctcaaac tatgaaacta ctaaaagaaa acacggggaa actctccagg 65880 acattgggtggggcaaatat ttcttgagta ataataccac acaagcacag gcaaccaaag 65940 taaaagtggacaaatggaat cacatcaagt taaaaaactg cttgcatggc aaaggaacaa 66000 tcaatgaagtgaagagacaa cacacagaat gggagaaaat atctgcaaac atctgacaag 66060 gtattaacaatcagaatata gaaggagctc aaacaactct acaaaaaaac ttaaaaatcc 66120 aatttaaaaatgggcaaaag agctgagtaa acatttctca aaagaagatg tacaaatggc 66180 aaatgggtatatgaaaagga gttcaacatc attaattatc agagaaatgc aaatcaaaac 66240 tacaatgagataccatctta ccccaaagta gcttatatcc aaaagatggg caataacaaa 66300 tgctagtgaggatgtagaga aaagggaacc ctggtatact gttggtcaga ttgtaaatta 66360 gtacaactactatggagaac agtttgaagt ttcctcaaaa aactacaaat agagctacca 66420 tatgatccagcaatcccact gctgggtatg cacccaaaag aaaggaaatc agtatatcga 66480 agagatgtctgcagtcccat gtttgttgca gcgctgttca caatagccaa gatttggaag 66540 caacctaagtgtccatccac aggtgaatat agataaagaa aatgtggaac atatacacca 66600 ctattcggccataaaatgaa tgagatcctg tcacttgcaa caacacagat gcaactggag 66660 gtcatgttaagtgaaataac cagacacaca aagacaaacc tcccatgttc tcacttattt 66720 gtgggagctaaaaataaaaa caattgcagt gcctcatgcc tgtaatccta gcactttggg 66780 aggtcgaggcaggcagattg cctgagctca ggagttcgag accagcttgg gcaacacggt 66840 gaaaccctgtctctactaaa atacaaaaaa ttagctgggt gtggcggcat gcgcctgtag 66900 tcccagctactcgggaggct gaggcaggag aattgcttga acccgggagg tggaggttgc 66960 agtgagtcgagatcatgcca ctgaactcca gcctgggtga cagagagaga ctccgtctcc 67020 aaaaaagaaaaagaaagaaa acaattgaac ttgtggggat aaagtagcag gttggttgcc 67080 agaggctaggaagggtagtg ggagtgggga aagtgggagt cccagctaca tgggaggctg 67140 agatgggaggattgcttaag ctcaggaggt ggaggttgca gtgagttgag atcacaccac 67200 tgcaactccagcctgggcaa cagagggaga ccctgtctcg gaaaaaaaaa aagatgataa 67260 atcaaagtattttaataaaa ttgggccata ctagagatgt tattgtttga aatcaaatat 67320 atgaagtatagttaataata taagtgtaat agaagaaaag gagccttaga aagcttgaaa 67380 aacatcgttgtacttcatat acatcttctt tgcttacatt ataatggaca atgctgcaac 67440 aaacatggaagtgcagatat ctctttgcaa catgagagat tcatgtagat ctaagagact 67500 gtgaagactgtttcaatatt ggagttacaa ttagcttttt aattacctct tctggccagc 67560 tgtggtggctcacgcctgta atcccagcac tttgggagac caaggcgggt ggatcacctg 67620 aggtcaggaattcgagacca gactggccaa cacggccaaa ccccgtctct actaaaaata 67680 caaaaattagctgggcgtgg tggtgggtgc ctgaatccca gctatttggg aggctgaggc 67740 aggagtatcacttgaacccg gggggcagag gttgcagtga gtcgagatcg tgccactgcg 67800 ctctaacctaggcaacagag gaaggttctg tctcaaaaaa aaaaaaatta ccttttcatt 67860 gtttgctaatgtgtagaatt ctgcatgtaa catgtccagt ttaaagaatg attatagagc 67920 aaatccctgtgtaaccagcg ctcaaacaat gaaatagaat ataatggcag cccagaatcc 67980 ctttgggtggtccctcctgc cacacctact tccctccctg cagaggtggg acagtcctcc 68040 tttctgccttccttgctgtg tatggtttta ccacctacac gtgcatccct aaacgatgca 68100 ggttgattttctctgttttt gaactttatg tgttcatgtc ttctacatat attttgtgac 68160 ttttttcttttatgacttgc tcatttcatt tatcattgag attcatttat tctccgcact 68220 gaaattctggttcattatag gcttaatgta agatgttgag gccatattgt ttattagaaa 68280 ggcactaaaatgccctattc actttcactt ttgcttctca tctattttat tataatttct 68340 atttcttaaccctttctcaa acccatgtag ctgtcctcat cctccctacc aacaccccat 68400 ccagacatctctcatttgca caactacagc aggctgtcat ctggtctcct ggcctcatca 68460 gttttgctgcctctgattgg ttcttcgtac tgcatctgaa ataatttttg aaatataaat 68520 attcttagctcttccttgca taagagaatt aaaagtacca ttgtcttaga gattgctata 68580 taactaacaagttcaaactc ttaggatttt actacaatgg gccttatttt ctgtacttct 68640 gtgtctttgagttgtttaga gtagctctac ttcataaact tggttctggg cttttgtggg 68700 aggtcaggtctgttccacat gtccccacgt tcttcttgga ccagtggcta cctcacagga 68760 gctcaagaggccaagccaaa ctgttggagc agcacatcta ttgatatatc attggctata 68820 aaaagtcctgtggtcaagcc caacatcaac tggtagggaa gtgtttgctc tctgcgcact 68880 ctagtacactgcagggtcgc aaggctgagg gagagaatga agaattgaga acggtaatcc 68940 accacaactcttgtcaatag cagtactttc tgtcattatt tagattgcta atttctttat 69000 ttgttccttttgttatttta tttgactatg aattcccata aaaatattgt attaaacccg 69060 aaagagggatatatgtaaaa gaatataaga agttgaattt gatgacttga tttacaactc 69120 ttgagttctgtgacttggag caaatcaatt taatgttagt cttattttcc acatccaaag 69180 gatatatttttatatctctc ttttgagaat tctaagaata tgcagagaat aacatattag 69240 taaaaaaccaggatattgaa atgttcctag gtctccttta ctcattaaca aggtgacaat 69300 gtagcttgactttggctttg tacctgtact ggtcattaag aagatgtccc ctatctctca 69360 gctggaaagtgttatcagtg ttgttgacca ggaagagatt taactaagag atcatagcaa 69420 taatctttttttccctccca ctctgctata ggacataatg attatatttg tccagctaca 69480 aatcagtgtacaatcgataa aaaccggcgc aagagctgcc aggcctgccg acttcggaag 69540 tgttacgaagtgggaatggt gaagtgtggt gagtgcttgc ttcccttctt attgaatatg 69600 ggccttgctaaaagccctgt cctctgagga actggggaca ggtagccggg aaaagagaag 69660 atttgggacatagtaattaa gtatttgcgt gttgtcacat tggagggggc attgacttat 69720 ccacagtaactgcagaggac agagctgggg tgaatgggaa cagattatgg gaggcagatt 69780 ttggccccaggtagagaaga gctttctaga gttcaagtgg tctgaccaca gaataggcca 69840 ccagatgggttaggggactt ctagccactg gaatcctcaa acagggctgg gtggccgtct 69900 gtctgtgatcttgaaaagtc cagttctaag gatgaagtcg tggtaaatgt ccatggttaa 69960 aactcgtgacaaaaaagtag gatatcttgt gggttactgg ggtagccatg gggaggctca 70020 cacctatcccctcgtctagc tttctagaag tagaaaaata tgtaggagtc agaaacataa 70080 tggaactatgaaaagtacat acagcataga ttttgttcta tgacagtcat aggtgtatac 70140 atatgtgtatttaacatatt cacatacata tattcacatg tattttgtac actcacatac 70200 attcgcatatattttatgca caaagaaaag tgagcacttt ggtatataac tgacaaagat 70260 gccaacacccagctctctcc acctggctag attttggttc acttgtctgt actcacttgc 70320 ttgtttatatgtactcagag cagttctgcc tgcacttatt cttctgctag ccagtatttt 70380 acctgtggttaattgtaatt tctctgtgta attatttgaa aatttaaaac aaaaatccat 70440 cactttactctctgacaatt tctttttttt tttttttttg agatggagtc tcgctctgtc 70500 acccagactggagtgcagtg gcccattctc agcccactac aagctctgcc tcccgggttc 70560 acatcattctcctgcctcag cctcccaagt agctgggact acaggcaccc accaccacgc 70620 ctggctaatttttttttttt ttgtattttt agtagagatg gggtttcatc atgttagcca 70680 ggatggtctcgatctcctga cctcgtgatc tgcctgcctt ggcctcccag agtgctggga 70740 ttacaggtgtgagccactgt acccagccct ccctgacaat ttcttagtag ctttgccttg 70800 tgagcattctctgccctttt cttttctctg tgtatgtaac agattagaac cctcagctat 70860 tatagttcagttacagcaga agttctcttc atctgatcat gcttctctgg cttcctagag 70920 tcactgatgatcttcatttc ctctgtagaa catcctgcca gtgcccatag cctcacagcg 70980 tgtattattggttattctct caaacaccta aacatttcca ttcccaccgc ttcacattat 71040 ccttgtcagaaaccggtggg cttcttttca aacctgtttc tactcactgt aattgttaca 71100 ttataaaatttaattaaaat ttactcaaac atattatgaa taggaaaaga caagtttggt 71160 ttttttctgtgaaagtttag ttaaggccgg gtgcggtggc tcacgcctgt aatcccagca 71220 ctttgggaggccaaggcatg caaatcatga ggtcaagaga tcaagaccat cctggtcaac 71280 agggtgaaaccttgtctcca ctaaaaatac aaaaattagc cgggcgtggt ggcatgtgcc 71340 tgtagtcccagctacttggg aggctgagga aggagaatag tttgaacctg ggaggtggag 71400 cttgcagtgagccgagatcg cgccactaca ctccagcctg gtgacagagt gagactctgt 71460 ctgggggcggggggaggagg aagtttagtt gaaagttttg aataaaatct taaaggacta 71520 atagctattgagataggtat gggtgagact gggggaaaaa aacccataaa ccttgggaga 71580 tcctgaattcagaattcttt agaagtatct aggttcttgc tctgtttttg ttttaaagag 71640 gctgaaactgaaaatccaga gataatatct tatgtgtatg tttatgcaga aaagtgactt 71700 tgtctaattggcccagatgc ttaaagagaa agccttggca ctctgacaaa agattgcaaa 71760 taaatgttttaagttttaag ttaaactatt ttaaagtgag tatgtgtgtg tgtttaaaaa 71820 atgatttccaagttagtctt aagaatgctt ttattatact aggatccgtt gcacagctat 71880 tgccctcatggctcaaggca gtgtatgcag ggaagagcat ggaggttgga tcccatagag 71940 tctatgtttcattttcgttt catcacttcc tgctgactgt aactgtgctc aaactactga 72000 atcacctctttggcccttgg ttttcatgtc tctgaacaga gatatctgct tcacttggtt 72060 ttgtgaacaataagtatgaa aacatatatg aagacttagc acaatatctg acactcaatt 72120 ttagttttccttccattctc tctccttccc tgaaaaactc atatgagctt tgatacaaca 72180 ctgtttcatgagacagagta cagagggata gttaaagaag ctttcataga aaagggaatg 72240 agagaaaggttgttgtattt agccagaaag tctaagaaaa gactgtattc tctttggaga 72300 ttatggaagaaatgagatgg gttgttgcac atatacaatg ggatattttg cccttcactg 72360 accatagagaaagatcatta gagatgaact ttcttaactc tgtctctctc ctttcccatc 72420 actcttcttatctgccttcc ccaaaaccct ctatgcatgc tttttcttct atcaggtttg 72480 gaggactagagattctacct gcttgttgga tcctcctgca ccatcctgct tcttttattt 72540 tgaaaccatgtagtctgtta tcaccctttt cttctgaatt tctgatcttg tcttttctac 72600 tgaagtatggatgtggtcat ataatggtag gacaacaccc acctagacta actttatgga 72660 tgaaacttcattataaggat atactgaaat gtaaggagcc aggaaatccc tctgaatagc 72720 catgtatttggcctatatcc ccatattggg acaatagctc aacatatttt gggtgccata 72780 tctttatatacctgctgtat actcttctgt gaaagggatt tgataggtgg gtagtataaa 72840 atagtggttaaaagcaccag ctctggattt aggctactgc ttgggtttag atcctgcttc 72900 tgctattttctagctgtgcc atcttagaca agttatttga gcttatgttt ggttcctctt 72960 ctgtacattggagacagtaa tagttcctgt actgtagggt agctgtcagg acatgtgcaa 73020 tatgcaatgcctggtgcata gaagcttcca gtagacatta gctgccattt agtgtcattt 73080 atcactacgatcatcatcat ctttggctgg ggctatttac cactgcctaa tatggagcac 73140 tcgatttggtggagctgctc attaagctca ctcagaggca gctgcctagg ttacagtgtc 73200 aaagccacaacactagaaac atccttgata gaaaaatgag ctccttgtca agggctttat 73260 tgagtctagaacccccagaa ttcactacag gacctagaat gttagacttt gtcagtgaaa 73320 atttgtcaagtaaatttgaa cgtatgaatt caaaatctct cactttgggt atgtaaaggg 73380 tatataaatctgttttgtaa attccttatc cttatatact ctatactcta caaaagagaa 73440 atgtatgatcagaaaggtgc tttttttttt ttcttttttt ttttttgaga cagggtatca 73500 ctctgctgcagcccaggctg gagtgcagtg gtgcaatctt ggcttactgc aacttttacc 73560 tcctcgggctcaattgattc tcccacctca gcctcccgag tagctgggac tacaggtgtg 73620 tgcaccaccatgcctggcta atttttgtat tttttgtaga gacaggattt cactatgttg 73680 cccaggctagtcttcaactc ctgggctcaa gtgatcccct tgcctcagcc tctcaaagtg 73740 ctgggggattacaggcatga gccaccttgc ctagcagaaa ggtgcttttt aaaactatac 73800 attttgcagcaaaccgccat ggcatgtgca tacctatgta acaaacctgc atgttctgca 73860 catgtatcccagaacctaaa gtatattaaa aaaattaaga aaaacataca ttttgctcca 73920 ttttatcctgggtgtataat tgaccttagc attctgcttg attactaata aaatgaattg 73980 tattttaggcctttaattct tttagcagta aatttggttc aaattttctg aataaataga 74040 gccctttcttctactataac tagtcaatgt taagaggaaa ttctgacaaa ttttcctggg 74100 agccaataatttaaatttgc tcacattttc taactaatat ttatttttaa aaatgtaaac 74160 aattgatttagtgaataaac ataatgatgg gtgtataaaa ccaagcattt tgcagatttc 74220 aacttttagggtttcttttt ttaagggaaa ttcatataaa agttataacc atgctaatga 74280 catccttacttacaacatgt ctttcttaat ttccatttta cattttttgt ctttaaccga 74340 tgaaaacttataaagatgtt ggtgctcaaa tgtataggga tttggaagtt atatttttgt 74400 tgttgatttccatttttctt atcgtcagag aatatgatct gaataatacc tattttaaga 74460 ttttcttcattgcctagcat gtgataattt ttgcaaaata tctatggcct ttgtagatca 74520 agcttgttaattatgtggtt caaatattct gcattctgac tttttgctct ttcagctgtt 74580 gagagagagatttaaatagc ccgttataat actgcatctg tcagtttctc tttttctttc 74640 agttactttttgtattgtgt ttggaggctg tgttttattg tttgtctatt tatttattta 74700 tttatttattttttcaaccc aagtcttgct ctgtcaccca ggctggagtg catggcacgg 74760 tctcggttcactgtgcctcc tgggtttgtg cgactctcct gcctcagcct cttgagtagc 74820 tgggactacaggaatgcacc accatgcctg ggtaattttt gtatttgtag tagagatggg 74880 gtttttccattttggccagg ctggtctcaa actcctgacc tcaggtgatc cgcccacctt 74940 ggcctcccaaattgctggga ttacaggcat gagccagcgc acctggcctt tgttgttttg 75000 aagtatatgagtttagaatt atttatcttt ttaaaatatt ctagcgatga gtctccttat 75060 ctataataataatttttgcc ttaaagttta tttgtctggt atcaatagag taatgtcaat 75120 ttatttggttaattttgcct gttaaatatt tttctatctg tctactttgt ttttctatat 75180 gttaggtatatctcttacac ctaatctatg tctagattta aaaatatgta taatttcaga 75240 gtcactcttaaatggtcaat ttggtatttt ttgtttattg tgataactga tatttgggtt 75300 catttctatcatcttatttt ttgatttaaa aaattttatt atgtattttc tacttcttcc 75360 cttttaggagttgatcacat ttttatgttt tctttttctt cttttgctag tttaaaagtc 75420 atacattctgtttcaattcc cttttatctt ttgagacaga gtctcgctgt gtcacccagg 75480 ctggagtgcagtggtgtgat cttggctcac tgcagcctct gcctcccagg ttcaagtgat 75540 tctcctgcctcaccttccta aacagctagg attacaggca tttgccacca tgcccagcta 75600 atttttgtattattagtaga gatggggttt caccatgctg cccaggttgg tcttgaactc 75660 ctggcctcaagtgatccgtc ccctcccgcc ccacccgccg aaaccacctt tggcctccta 75720 aagttctgggattacaagtg tgagccacca tgtttggcca tgtttcaatt cctttaatga 75780 cgtttatgttttgtaacgtg ttcttgatta atttgagaat ttaagcttct atcttcccaa 75840 aaaagaatcttagaaattct aaccaaaatc attcctctct gattttgcat gttattgttt 75900 gttattttggttccaccttg tttctatatc actaaaactt aattcttggg ccggatgtgg 75960 tggcttatgcctgtaatccc agcactttca gaggccaagg caggaggatc acttgagccc 76020 aggaattcgagaccagcctg ggcaacatgg tgagaccctg tctttacaaa aaatacaaaa 76080 attagtcagatgtggtggtg cacacttgta gtcccagcta tccaggaggc tgaggtggga 76140 ggatctcttgagcctgggag gttgaggctg cagtgagctg tgatcatgac tgtaccacca 76200 tacttcagcctgggtgacag agaccttgtc tcttaaaaaa aaaaaaaaaa aagtaaatcc 76260 aaagaacaagcatataaatc aactttgcta gtaattaaaa catacaaagt aaagcgagat 76320 ggtttagttagattagcaaa cattaaaaat gatttttaat gcccaatggg ttctgagaaa 76380 acagtcaaactattgaggac tgggtaacat agtaagaccc tagttctaca aaaaaattta 76440 aaagttagctgggcatggtg gcatattcct gtagtcccag ctactcagga ggctgaggca 76500 ggaggattgcttgagtccag gagatgaagg ctgcagtgag ctatgattgc atcattacac 76560 tccagcttgggcaacagagc aggactctgt ctcaaaaata caattaaaat agtgtagata 76620 ctacaatctaattttgtgta taaaggctgg gtgcagtggc tcacgcctgt aatcccagca 76680 ctttgggaggccaagatggg cagatcactt gagaatcagg aatttgagag cagcctggcc 76740 aacatggtgaaatcacatct ctactaaaaa tataaaaatt agccaggcat ggtggcgggc 76800 tcctgtaatcccagctactt gggaggctaa ggcaggagaa tcgcttgaac ccgggaggct 76860 gaagttgcagtgagccaaga atgtgccact gaactgcagc ctgggtgaca gagtgagact 76920 ccgtctcaaaaataaataaa taattttgtg tataaaacgt ggaaaaatat ggtggcaggc 76980 acccatagtcctagctactc gggaggctga ggcaggagaa tggcgtgaac ccggtaggcg 77040 gagcttgcagtgagccgaga tcacaccact gcactccagt ctaggccaca gagcaaggct 77100 ccgtctcaaaaaaaaaaaaa aaagaaaaaa gaaaaataaa agcattaaaa agactgaaag 77160 agtttatgccaaaatttatt ctcttctata tttttcagat tttttcactt aatttgttat 77220 ttgaaatatacttgttttgt gtaagtataa ggaaatatat acatatgcac acatgcatat 77280 aaacattttaagaatgtgtt ataataaaag tatattattt gatacctttg gaaatatccc 77340 catttttctacctgaagaaa attcctaatt tcatggtttg gaaacaggtt tatgagcact 77400 ctttatagagaaacggtgtt agtatctata gatgacctgg aaatggagac ctaaaaagtt 77460 tctgaaaagttatgtcgttg gttttgctag tacggtcacg accatagtaa tctttggtac 77520 gtgccccacaggctccagaa aataaaagtc aagctgcttt tgcttgactg cggttttacc 77580 ctggcaattcgaatgactct gctttcctct tcaggctccc ggagagagag atgtgggtac 77640 cgccttgtgcggagacagag aagtgccgac gagcagctgc actgtgccgg caaggccaag 77700 agaagtggcggccacgcgcc ccgagtgcgg gagctgctgc tggacgccct gagccccgag 77760 cagctagtgctcaccctcct ggaggctgag ccgccccatg tgctgatcag ccgccccagt 77820 gcgcccttcaccgaggcctc catgatgatg tccctgacca agttggccga caaggagttg 77880 gtacacatgatcagctgggc caagaagatt cccggtaggg ctttctggct atcagttttc 77940 catgtacttgtagaaaggcc ggccgctaat atttaagggg caagagtaca aagtagaggt 78000 ccatgagctgtgcctagata tttaacaggt cctcagctgg atttgtaact tttaagtgca 78060 atatgttccttccttctgtc ttggcatacc taccttcaac aaggccgtgt tctgatttag 78120 aattctgagactcttctgag ttctgtaccc aacatggtag tgcagaaaga gttgtgcgtg 78180 gcccagccatttctattctt gactgccttc ttttcccatg gctagatgca tcccatacca 78240 ccttgcacaaaccctatcct gtgtgtccac atctgctaca gacactcacc tgttggccac 78300 ctctcatgcctagaggtggt ctgggaggat ggacccaggg aacctaccta ggctctggaa 78360 ttgggcttggggtcatttgg gcaagaatcc tagagtcctg gaacctggaa cgtggttaaa 78420 atgatagactccacattgac ccatttcttg gctgtggatt cctcaccttg aaaggagggg 78480 tggggtagagtacagtatga ctagtttgaa agtgaaaggt ttgtcagatg ctaaatagaa 78540 ttttgtaaattattgttcca gtagagaatc aatattatgt acataaatga atatgtatgg 78600 acaaacagagtaaatcagtg gttgaagtta cacgaatcat caatgggccc ataaacctgg 78660 aatgccatcaagttaaaaat gagcttagtt actcatgagt tgtcacttgg aacctgcgtt 78720 ttccatcctccaaagtgatc acttctctca agcccatttg taatatatat ctgaagtgct 78780 gtatgatgctaaaattacca gctaattatc atttgacttg gtgtttctgt ggaggagtga 78840 atctaggattctaacctaga gtggcaacac cccacgatcc ccctgtgaca gcttctccat 78900 gctgttctttacagtccttg aagaaatgaa gtctcttata agttctgagc cactgggggc 78960 attcccatggcctggagggc agcgactgca ctgggcaagc tgtaaagatg aggaggggtg 79020 agaagctgggggaagagaag ttttgggtaa agagcctggg gaactgaggc ctatggtgac 79080 agtatcatttggggactttt gttggcctgg gcccatttct tctgagcttc ctgaggattt 79140 ttggtttctagttgtatttt gttttgtcta gcatcttcac ctttgccaga attattttat 79200 tttctctgctttttcccagg ggaggcaata ctgatgcact ttcctctagt ttttgcttta 79260 aatgtattccaaacacgatt ttgcaggacc acacatggag agcagtggtg aaattaatta 79320 ttgctgaaagctgtgcacct tctttgtgcc ataagaaatc tgaactctta aactgcatta 79380 ttccttattcaagcctggtg ttttgaaaag ttttcaggaa acgtagacat aatctgaagg 79440 cgtgattttttttctcctct cttagctggc atagtcattg tccaaaccaa aaaatatata 79500 ttaaaatatcatctagcctt gatcttgttg aatatctaca agattaagaa ccgtgatctc 79560 tcttgggtaggcttattgtc aatcactatg ggtgagactg ggaaggtata tacacattag 79620 gaacctaaactgagcaaagc atgtggattt agaaagtatt tatccatctt tacattcata 79680 acaccattacattctccttg aggcagattt gcgttataat tgttcaaaga cttgaaccat 79740 gtgtgttctctctgctgtag tttcctcatc tgtaaaacaa gaatgataag agatcctgcc 79800 tataagacattctcagagat aggcattgtt acccccattt tcctataaga aaaacaaaga 79860 cttaatgggagattaagtga acagctagaa agaggctgag ctggggttcg aaccagagtc 79920 catttcactccaaggcggtg tcttttgtta tcatatttat attacatggc cctctctttt 79980 tatcatggcttgtgaaggaa gccccggtgt tctctgcttt gcttttgaag tgcttccctc 80040 cccagagattacctgtttgc aaacagtact gtgaccaaca tgggttatta ggttgtcagg 80100 acctgcttcgttattatatt tgctctttat ttatttattt atttatttat ttttgagaca 80160 gggtctcgctctgttgccca ggctggagtg cagtggcgtg atctcagctc actacagcct 80220 cgacctcctgggctcaggcg atcatcccac ttcagcctcc agagtatctg ggactacagg 80280 cacctgccaccatgaccaga taattttctg tagagatggg gtttctccat gttggccagg 80340 ctggtctcaaactcctgggt gcaggcaatc cacccacctt gacctcccaa agtgctggga 80400 ttacaggtgtgcttggctat atttgctgtt taggatagaa tcacccagaa acagtgcttc 80460 tacccagaagaaggatctta acactggata ggaaatttta atcaatcaga gaaatccttg 80520 cagttgaggccttggttttc tgtgagggct ggcactgctc tctgcaagcc tccaacccca 80580 acctccacctaccccatccc ccacctaccc catcccccac cccttctgat cccagtcaag 80640 gattgggtcagacaggcagg tcttctgact ggcagccaag catcaacatt ctcagtagtg 80700 cagaggaattatcaggacac agctaacaaa gatcagttct gagccgaggt cgtagtgctt 80760 gacaaactctaaatgaagta tatttgtctc tagaaggggt ccaagactgg aaactaagtt 80820 gcgcagcttaacttcaaagt tttcttcctt taatgagcag ttaatcacat ctataaaata 80880 tcaactccctaatggtttgt gttttcttag tgttttaaca cttgccattc tgtctctaca 80940 cacacagggagctgaggagg aggggtgggg gtgtctcacc gcctcttgct ttccccaggc 81000 tttgtggagctcagcctgtt cgaccaagtg cggctcttgg agagctgttg gatggaggtg 81060 ttaatgatggggctgatgtg gcgctcaatt gaccaccccg gcaagctcat ctttgctcca 81120 gatcttgttctggacaggtg agaaaaaata cattgtgttt cttctctgac ttgtttgagt 81180 aaggtgcttagtgagtggga acaaagtcct gggtgctgca attaaaatct cacacttgca 81240 gggcagaggatgatagcatc atcagctcct tcactgggtc aagaaccaga gaaggagaga 81300 gttgggtccaaggattcagg gtcctgtgac tcatttttaa tctgtggtgc agcagcattt 81360 acaggccagcgctttaatag gggactgtat cccgtaggta tgtggccact atgtgtataa 81420 gtcgacacagatttttctcc attaaaaatt ccattttcag gttataatct taagttgtcc 81480 tgctgttttttgtacctata gtgaccaatt atatctggag ctttctggac aggtgataaa 81540 attcttagaaatgtgccaag tttattttca catgctttaa ctcactcttt tgtttttttt 81600 tgttttgttttgttttgttt tttgtttttt ttctgagatg gagtctctct ctgttgctca 81660 ggctggagtgcagaggtgca atcttggctc actgcaacct ccgcctaccg gattcaagtg 81720 atcctgctgcctcagcctct caagtagttg ggatcacagg tgtccaccac catgccaggc 81780 taatttttctatttttagta gagaagtggt ttcaccatgt tggccaggct ggtcttgaac 81840 tcctgacctcaggcgatctg cccacctcag cttcccaaag cgctgggatt acaggcgtga 81900 gccaccatgcccgatctgct ttaacacatt ctaatgcatg tactatatag catttttggc 81960 aatagcggtggaaggaaggg ttactaaaac tatatgaaac ttaacagaaa atgggacatg 82020 atgctgtatcttggttgtgt ttgattttct tttaaagatg acacagaaaa ggaaacaatt 82080 tttaattgacttaggtgaac tgtttatgga gggaaagctg gactgtataa aaatactcaa 82140 gctttttagcaggaaagtag aacaccctct tggtgtaaat tcgagcagtt cgaaatcttc 82200 ttggaaattgatttccacat ctcttttatg gaaaaagtgc taggttgaat gttcagccac 82260 atctgactctgcatagcgtg ggaggatgcc tagtgtctac cccaactctt gcattataat 82320 cctgttaccactttagatca tcagaagacc ctgtgttaca cagatgaaga gtgatgcccc 82380 aaggtatcagtccccattct gccttttgtc atggttgaca atgttattaa aagagcactg 82440 ttctgcataatggtgttttg atagagaaca gatcctctga gaagagctgg aggactgatg 82500 tgacttgaacaggagcaagc ccaggtggta aaccatggag ggaggctctg gaagaccaga 82560 gaagttcagggcacaagacc cttcagtaac aaacaaaata gttaacctat tggcttgtat 82620 gtgcttggcagcaccttatg catttaactt atgtcaacac atttaatctt cacaatcttc 82680 ctgccccctttgagggagta ggatccatta ttatctctat cattcagata ttggaaatgg 82740 gagattgagaaacctgctta caggtaggat aataggtggt ggagctggac ttggggggtt 82800 gccaaatggcaaactaactc tctactttat tctacctgtt gttatgggtg acaatgttga 82860 caaagagcacattctgcaga acagagatgt tttggtagag aacagccctg ttttacttgt 82920 aacacactgcagaaacccac tctccccact gtcatctcag ggtaccatgt cgcaaggcag 82980 gctgaaaagccaagcaccta gccaagccat tgctctcatt cattcattgt attctgcttg 83040 gtgttttaactggggccaaa tatacatatg tataaatata cacatataat tttccttgaa 83100 gttagtcctaggaacacatt ccatcccttg acaaataatt tgcagacttt aggattattt 83160 tatcttttgtcttgatttct aaattgatgc caaatttagt gtttattttt ggtgactatt 83220 tcattcctggtttttagtac aattaactct ccactctccc atttctctgt atgcgttctt 83280 taattcctgtaattgtgtgt atacattact ataagtggac acaaatcctg gaaaaatatt 83340 aggcctaccttttagttaat agaagaaaag ttatttttct tacaaattat ttctaataga 83400 cttacactgcctttataact taagtgaaag tattatgttg taaaacataa atctagtata 83460 tttgattgagtatagaagag gaattcttgg gaattgtaaa tgcattcatg ttgagcaggc 83520 atttttttttttttttggaa tgactactgg tgtttatttg ttgttgcaat ttctagtagt 83580 ttttgtttgtttgttttttg tttttgagat ggagtctcgc tctgtcaccc aggctggaat 83640 acgatggcatgatctcagct cactgcaatc tccgcctccc aagctcaagt gattcttgtg 83700 cctcagcctcctgagtaggt gggattacag gcatgtgcca ctacggctgg ccaatttttg 83760 tattttttttttttttttag tggagacggg gttttaccat gttggccaag ctggtctcga 83820 ggtcctgacttcaagtgatc ccccagcctc agcctcccaa attgttggga ttacagacgt 83880 gagtcaccacgcccagccta cagtctctag tatttttaac acattaactt tctgaagtct 83940 ggaacttgaagtctaagata gttcagttac ttagtcctct cttatacaaa tgaatatact 84000 tttatgtaataggtatattt gtagaggagt tgctcattca aaaagtcagg agtcatgctc 84060 cataaagacttctattacga ctcttttttg caaagtgaag ggaatcttca caccatttga 84120 aaataactgtcttctgctgg attgtcctag cagagcttct tcaagtggta atatggctga 84180 ataaacagtgaatacaacta acagttgccc atttgtggat actgaaacta taatttctgt 84240 ttccctttattcttgttgag gtgtccacaa caagaaaact tgtgtctact gaggatgaga 84300 ggaaaatctcattacttcag cttatttcta agcatttagt ttttctttta ctaaccacta 84360 aattcatcataaattcacgt gaagatctaa agaacctgac tgtctaattg ctcaaaaaaa 84420 agtcacatatgcaaagacat ttttgtgtcc ttagtatcaa caggcaactg actaatgtta 84480 aattattagtcagaggaagt ttgtatctgg cttggatccc attgtggaca tttgcagata 84540 ggtccgtggaattgtatatg tataaatgtc ttgagtttac attcacatta gttatttgta 84600 tgctaaattccttcaagata accaccgaat tttcaattcc caattctaag ccttaaacac 84660 tccctgccattgccatacac acagaggtaa accatggtct gtacccaggt gtgtgctgcg 84720 agcagagatatatatatata tatacacaca catacataca cacacacaca cacacacaca 84780 cacacacacacacacacaca caaatagtgt acccctaagg gaggcccact cattcaacat 84840 tttgttgttgtattaaacaa tattcttctt taggccaggc acggtggctc acgcctgtaa 84900 tcccagcacttggggagact gagatgggtg gatcacctga ggtcaggagt tcgagacaag 84960 cctgagcaacatgatgaaac cccttcttta ctaaaaatac aaaaattagc tgggtgtggt 85020 ggcaggcgcctgtaatccca gctacttggg aagctgaggc aggagaattg cttgaaccca 85080 ggaagtggaggttgcagtga gccaagatca cgtcattgca ctccagctgg ggcgacagag 85140 caagactccatcttaaaaaa aataaaaaat aaaaagcaat attcttattt tataaagagt 85200 gattattggccgggctcggt tgctcacacc tgtagtccca gcactttggg aggctgaggt 85260 gagtggatcacttgaggtca ggagttcaag accagcctgg ccaacatggt gaaacccctt 85320 ctctactaaaaatgcaaaaa ttagccaggc atagtggtgt gtgcctgtaa tcccagctac 85380 atgggaggctgaggcaggag aatcacttga acctaggagg aggaggttgc agagagcaga 85440 gatcatgccactgcactcca gtctgggcat cggggtgaga ccctgtctca aaaaaaaaaa 85500 gtgattgtcaagtaataaat ttgatatggt ttggctctgt gtccccagca aatctcatct 85560 gaaattgtaatctccacgtg tcaagggagg gatctggtgg gagtgattgg atcatgggga 85620 tggtttcccccatgctgttc tcatgagagt gagtgagttc tcacaggagc ttatgcttta 85680 aaagtgtttggcagctcccg gctgtcttgc tcagtcactc gctctcctgc ctccatgtaa 85740 gatgtgccttggtttccctt tgctctctgc catgattgta agtttcctga ggcctcccca 85800 gccatgcagaactgtgagtc agttaaacct tttttcttca tagattaccc agtctcagat 85860 agtgctttatagcaatgtga aaatggacta atacaaagat attccatgtt attactgatt 85920 ttatttagtagtttatggac agatagtgtg caaaaataaa tttcctgagt aggtcagttt 85980 ggttgatacattgtttcaat attttaacat tcaaatcata tgccctgttt ttgtttttgt 86040 ttttttttttttagagacgg tcccgctctg tggcccaggc tggagtgcag tggtgccatc 86100 acggctcactgcaaccttgg gctcctggcc tcaagtgatc ttttgcttca gccccctgag 86160 gaactgggactataggtgta tgctaccatg cctggtttat tattattttg tagagacaag 86220 gtcttgctacatcgcccagg ctggtctaga actcttggcc tcaagtgatc ctcccacttt 86280 ggcctccccaaagcacgagg attacagaca tgggccactt tacccagcca gccctgtctt 86340 taattcaactcttttaaccc tgtcctaatt tcttactcat aattcagttt caatctaaaa 86400 ttataaaataaataaaatag atgtcattaa tttagagtct ttactaactt tgttctgtgt 86460 aactcatctgaaagaccttt actgggctgt catttacgat gtcttatact ttattgcctc 86520 ttcatttctcatttattttg ttgaaatgta tttggtttct gcagactgtg gagtgaataa 86580 aaatattctaactttgccac cccttgaatg aaatggttga ctgtcacacc tgcttaaaaa 86640 gaaagcaatcagacctagtt ctttagactt tgtttagaaa ttaactttct ccatgagtta 86700 tgtatggtctgattactgtg agggacagcc tttatcaggg tttaaaatac ctcagtatgc 86760 caaccctcctcccatttttg gaacataaat ttgcagtgaa aatggcatat attttaatga 86820 ggaaatgataccaatttcaa tattgaggaa ctaaggcaca ggtacttctg gaaaatagga 86880 ttgatttcaggtgggttccc ttaccatacc acttggggag gggtgtgtgt gagtgtgtgt 86940 gtgtgtgtgtatgtattgtg catgtgtgta taatcccaca tcagcacaga agaataaaga 87000 gataatcaaatatcaatgca ggagttggtg ggtttttttg tttttttttt tttttgcccc 87060 atagagatattttcaaacta gctttcctta gtatcaaatg tccccaagtc caacagttac 87120 aatttccaataattaattgt ccgcaggcaa ggtgattcag gtgttttttg tgttatctct 87180 gtgcagggcttgtgttgtcc ttactggatg cctgcatcag gttgcctggg agagcctaga 87240 gctggggaggtggaaagatg aggcttcctg tagatttggc actctttgcc cagtgctctg 87300 gattctctaagacggccttt tcctatgagt gacttccagg gggcactggt gttttgtcac 87360 ttaacctgtgtacttataga aaattgcagg tgtttacaga atttatgatt tagtaaattt 87420 agtaactcagtaatgctcat ataccaaagt gagcaatttg catgcttgta gctctgtgtg 87480 agcgagtctgggtgggagag tgtgagtgct tcggaatgca ggatcccggt gagtgccatg 87540 tacggcaggtaatgggaaag acttctgcag gactggtgta tccagtggtg tcagaggctc 87600 ttccctgaaatactgccatc gctggaaatg ccctgagttc ggggaaggag gaggggagca 87660 gccagctctttgaagacctc aaggcccctt caggggctgc tagagactaa aaatggaact 87720 cgcataaacccactgccctt tctgtgtgct gcaggctttt gggagcaaag ggtggttttg 87780 tgacaaaatcatctaactgc ttgtcaagga cttccaataa ccctgtgact gacaataata 87840 gagtgttttgggggagcagt gaggtggaat aatgtgtgtc tggctggagt gaatagaagc 87900 tggtattttccagataaagt tcaagtaatt tacttccaaa gtatatttaa acatttattt 87960 ctacaaggagtgctccaaag aattttgatt agatggctca aagtttaaag ataatccttg 88020 cttgaagataatccttggca agtcaaaaat ttttccccac ctccatgtat accttctttt 88080 cctgattctaatccatcttc tctaattgcg atttctttct catagtcagc tttttcaaat 88140 tacaggtaaatgtcttagtt gctacacaag tttctaagtg accaccagga agtgagagtt 88200 aagccctagatatggagttt tattcttggg atatttgctt ctgtgacaca cggtcttcct 88260 cattaatacttcccgatggg aacatgaagt gtctcatttt gaaatacgtg tcatatctgg 88320 ggctggttgactgatatggt tttgattgaa aacaatcaat aggagtggta ttgctgtaag 88380 aaaacatttggtgaaaatgt agaaggaaaa tattccaatg cacatttttg cctaaataat 88440 atttattcatatatttactt cagggattta taggaaatgg cctatcttct ttatatgaag 88500 acaattctagtaatttcata ttgctgggtg tggtctcatt aacaccctgt tgtagttaaa 88560 atgatattatcagatgaaca tgttacaaga tgaaacttga gattaaaaat aaaacattcc 88620 ttattgtttttttgatggtt tcctgaagct atgttcctta aatttccaaa cgaacttttg 88680 tagggatgaggggaaatgcg tagaaggaat tctggaaatc tttgacatgc tcctggcaac 88740 tacttcaaggtttcgagagt taaaactcca acacaaagaa tatctctgtg tcaaggccat 88800 gatcctgctcaattccagta agtaatcaca cagctgggcc atgttttatc ggggagagat 88860 gctgtttctacaactagcgt gatattaaga agaatgttga acttctattt tatttgaaag 88920 ggtaaaatggtttccttttg gacttcgttt ttattttgat agcgatttaa actgtaggta 88980 acttttggtaacttggacat aaattactca ttaagtgaat gactggcaat caatttaaaa 89040 gtagctcaagccacttgctg gaaaagaaaa aaaaaggaac tttaaattgt ttatctttta 89100 aacttttttcagtgctcaca cagacacttt acatggttgg catgcattta tacttatgtc 89160 tggggtcctccttttttaca gattcattcg ttcagtaaag atacaatcct accctcaaat 89220 ggctcatagtttaggcaggg agagagagaa aacaaatcat taaaaataat gatttctgtg 89280 ctatgataaagtctacacaa aatactacgg gaaaatagga ggagagatgc tggagttgtt 89340 gcagaagggaatgattgaac aaatcttcag gaaagagcag agggaagtag gtatgacttt 89400 aaaatgcagtgctgaagatt agaaactgct gcccaggctt tgggcagctt agaagaggtt 89460 caggcaggggagtgtcatgc acagatatgc gatatagaaa ggtcactctg gcttccatgt 89520 ggaggactagaaagggcaga gactgaagcc gggggcccat tagaggcaat gagagcctga 89580 actgacattatggcgtgagg tcagggagca aaggacttga cttgaaggaa aagtgggagg 89640 tagaggagggaaataaggtg tctaggatat gcagatggtt tcgttttgtt ggttttatct 89700 tatataaatatctgattatt gttaataaac attcaaatga gaaaaacata caaggaagaa 89760 aataaaatcatcaggaatac ctgccctcaa aataaccacc ataactttgg tgaccattcc 89820 tttctttctttttttttttc tttttttttt tttttttgag acaggatctt gctctgctgc 89880 ccaggctggagggcagtggc atgatcatag ctcactgtaa cctagtactg agctcaagtg 89940 atcctcccaccttggcctca caagtagctt ggaatacagg tgcataccac cagacctggt 90000 taattaaaacaatttttttt tgtagagaca gaatcttgct gtgttgccag aggtggcctt 90060 gaactcctggcctcaagcag tcctcccacc tcagcctccc aaagttctgg aatttacaag 90120 cgtgagccactgtgcacagt ctgtatcttg ttttttcact tttctttttg agacagggtg 90180 tcactctgttgcccaggctg gaatgcagtg gcacgatcat ggtttactgc agttccgacc 90240 tcctgggctcaagtgattct cccacctcag ccacctgagt agctgagacc acaagcacct 90300 gccaccacacccgaataatt tttgtatttt ttgtagaggt gaggtatccc tgtgttggcc 90360 aagctggtctcaaactcctg tatttttgtt tttcttttca aaatgctgtg aaaccattct 90420 gggtcaactaggagagatct aacacaatct ttgatatgag ggcattatac taaattgttc 90480 aaccatttctctgttattaa atatccagtt cctcttcctt tttaaccatt ataaacatta 90540 ctgcaataaatagagatgtg ttattttgta tgaatttcta agtttctgga tggttgtcaa 90600 gactggtcatttcacgatct acctggtgtc taagccagcc gctctagcag atattgatgg 90660 ctttgcttagccatttactc ttgtcgagcc tttaggttat tgactttttt tcttcctcaa 90720 acactgtatatccaggtttt aatgttcacc tgaagactta cagatatctc tatttagaca 90780 acatattgggccttatttat ccaatcttag agttcgatac ttgaaacaac agggatatat 90840 cagatcatattatactacgg tctttaaatc agccaaagta gcagttcctg aagccaagat 90900 tcaatgcagaattcactgtg gtcacatgtt tccagctgcc tcttgatctg gggccagctg 90960 accttcatacgtgtcttctc tcacagactc ttggatattg gcactagttt ttttgtttgt 91020 ttgtttgttttgagacagag cctcactctg ttgaccaggc tggagtgtta aggatagtta 91080 cttttgcaaaatataacaaa aatgaattat gagaaaaata aaagtgtaaa atacaagtcc 91140 tagtttataatattattagt tatcacattc aactgattta aaattactct gtcgattgct 91200 aaaaatgttcctaaatgctt acactcaatt tctacccatc tctttgtaaa tgggcaacag 91260 accatacattagctctggag agagcacagg atactgtcca agagttgctt ggatctaggg 91320 tggagggtggggttagcccc tgaagaactg ggtgagggag aatgaagaag gaatctgaag 91380 gccatgtgaaggtacagaga cctggaagac aacctttgag catttcctag ttaagtctca 91440 atctggccttacctgcctaa caggtcattt cccctgcacc caacacaccc ttccctgttt 91500 attgttaccattcacctttt acagaataac atgagggccc agcttatact gatgattatg 91560 ttgatgtgcatataaggaaa gtccagccag gtgtgttttt tttttttttt tttttttttt 91620 ttttttttgacagagtcttc ctgtgttggc caggctagag tggagtgcag tggtggaatc 91680 ttggctcactgaaacctccg cctcccaggt tcaagtgatt ctcaagcgat gcctcagcat 91740 cccaagtagctgggattaca ggtgcatgcc accatgcctg gctaattttt gtatttttag 91800 tagagatgatatattagtct gttctcacac tattatgaag aaatacctga gactgggtaa 91860 tttataaaggaaagagattt aattgactca cagttcagta tggctgggga ggcctcagga 91920 gacttacaataatggcggaa agtgaagagg aagcaagata ccttcttcac aaggtgccag 91980 gaaggagaagtcccaagcaa aggcagaaga gccccttata taaccatcac atcttgggag 92040 agctcactcactgtcatgag aacagcatgg gggaaactgc ccccatgatt caattacctc 92100 cacctggtctctctcttgac acatggggat tatggagatt acaattcaag atgagatttg 92160 ggtggggacacaaagcctaa ccatgtcaga cagggtttca ccatgttgtc caggctggtc 92220 ttgaactcctggcttcgagt gatctgccca ccttggcctc ccaaagtgcc gtgattacag 92280 gtgtgagccaccacatccgg cccaccagcc aggatatttg aaattgatca tggaataaga 92340 tcaaccctttctgacctttt ccaaaccacc taccaacatt acctcacata ggtgctgcca 92400 tttctgtcaaagggaggatc tgcttgaaga gtaccttccc atcttggcaa tggaagatca 92460 tcaaatgccagatgatgggg cttctctcac tttcagaaat aatttagatc tcttttctgt 92520 gcaggaaagtgcttctcgga aagcactgtt tgcttgttgt tacaacactt tacagtataa 92580 agccttctgtttggcaaggc tccttatagg cattttagcc tcccagacat catattgtgt 92640 tgttgtcaaagctagacgca gcatctgtgc aaatgggaaa gatgaaggct acagcattcc 92700 cctgcagcatgacagaactg ccacattgag ataattacag aaggcgaggg agacatgtga 92760 tgtaattaccacttgtggca gtaaacgagt aaaatttgtt tactgtaaat ccaagtttaa 92820 gaaatctttttttttttttt tttttttttt tttgacagag tcttgccctg tcgcccaggc 92880 tggagtgcaatggtgcgatc ttggctcact gcaacctcta cctcctggct taaaacagtt 92940 ctcctgcctcagcctcctga gtagctggga ttacaggcac ccaccaccac gcctggctaa 93000 tttttgtatttttagtagag atggggtttc accatgttgg ccaggctggt ctcgaactcc 93060 tgacctcgtgatccgcttgc ctcggcctcc caaagtgctg ggccctcgcc tggccagaga 93120 tcttcttttaaggaagttcc tttcttggta gtcataacaa ttgtcaaaat aaattgatcc 93180 tgttcagcattgctatggcg aaaatgggac aattttcact gctaggttaa gtgagtcttt 93240 tctatgctaggttttaagga tttgtaagta caggcttttt tcttctggat tatttgtggt 93300 atttaaatttaaaaaaaaat agggatggaa tctgcctccc cgccttaaaa tttaaaaccc 93360 tgacagaatatataaaacag atattggaca ttggacaaca gtgatcccca ggaggaggga 93420 cacaaacgaggagagccctt tgattgtcca gtttactgcc tggagccagt ttccaggttg 93480 caaagcagggatgggtgtgt taggtttctc cagagaaaca gaaccaatag gatggatagg 93540 taggtaggcagataaatgag aggggattta ttatgaaaac tgacttgaac aattatgaag 93600 gctgagaagtcacatgatat gcgtctgcat gctagtgaac cagggaagcc agtagcatgg 93660 ctcagtgtaaatggaaagac ctgagaacta gggagctggt ggtgtaaccc tcagtttgag 93720 attgaaggcctgagaaactg ggaggccact ggtgtgagtc ccagggtctg gaggctggag 93780 aacctggagttctgatgtcc aagggcagga gaaaatgggt gttccagctc cgagagagag 93840 aattctcttcctctgccatt tcgttctatc tgggcactca gccaattgga cggtgcctgc 93900 caacattggctaagggcaga tcttccttac ttagtccact gttctttctt tttttttttt 93960 tttgagatgaagtcttgctt tgttactcag gctggagtgc agtggtgcca tcttggctca 94020 ctgcaacctccaccttctgg gttcaagcga ttctcctgcc tcagcttcca gagtagctga 94080 gattacaggcatctgccacc acgcctggct aattttttgt atttttagta gagatggggt 94140 ttcaccacattagacaggct ggtctcgaac tcctgacctc aagtgatcca cccactttgg 94200 cctccgaaagtgcagggatt acaggtgtga gccactgtgc ctggccttag tccactgact 94260 ctaatgccagtctcttcctg gaacactctc acagacatac ccagaaataa tgctttatct 94320 gctatctgggtatcccttta tccagtcaag ttgacaccta agactaacca tcacaaaggg 94380 taacccaaatagacaccagt ggtctccctt ggtagcaagg cagctaggac ttggagggga 94440 gagtactgagtgggaaagag ctgcacaaag aattttggag atctatggag agtcctcttc 94500 aagtcttcagctgagtgcta atctgcccat gcttatgagg ataccaagga cagggaaaga 94560 accatcagaaaggagcgggc gaaacaatcc ctagagttca cacagggcca ggaacagttc 94620 acattctcaccagccagtgg gaaaaacctt gcagttcact gggtattggg cacttctcag 94680 ccttcctatagtattcagaa gggtattgcc tcagtagtgg gcctagacta aaagccatta 94740 tgatcctaccaaacaaaaaa gcaagcctgg aggatcaaac aattgctaag tgatttaact 94800 gcatcccagcacaaagctca agagtagaga cacatcccat ttcattacat gttgctttat 94860 tgtgcatcacagatactgca tgtttttaca aatcgaaggt ttgtggcaat gctgcattga 94920 acaagtctgttagtaccatt ttttccaaca gcatgtgctc actttatgtc tgtgtcaaat 94980 tttgataacactttgcaata tttctaactt tttcattata tctattacag tgatctgtaa 95040 tcagtgatttttgatgttac tattgtaatt gttttggggt gccacaaact atgcccatat 95100 aagctggcaaacttaaccta taaatttgtg tgttctgact gctccaccaa ctggtggccc 95160 caccaccatctggaattctg ggagaattct accatgcatt tgaggaagga ataataccaa 95220 gtgatatggtttggctctgt gtccccaccc aaatctcatc ttgtagtgcc cataattccc 95280 acatgttgtgggagggacct ggtgggagat gattgaatca tgggagcagg tctttactgt 95340 gctgttctcatgatagtgaa taaatctcac gagatttgat ggttatataa aaatgggagt 95400 ttccctgcacaagccctctt ctcttgtctg ccgccacatg agatgtgcct ttcaccttct 95460 gccatgattgtgaggcttcc ccagccatgt ggaactgtaa gtccaataaa cctttctttt 95520 gtatattgcccagtcttggg tatgtctatc agcagtgtga aaatggacta atacaccaag 95580 tttacacatactcttacaga aaattgaaca gcatggaatg ttttccaatt cattctgtga 95640 ggccagcattactctgatag aacactcaga ctaacacact agaagaaaag aagacaacag 95700 accaatttccctcatgcatg tataagcaaa agttctctaa attttttttt ttttggtaac 95760 tagaatccaaaactgtatta aaagaatagc acatcatgaa caagcagaat ttttgggaat 95820 acaaggtttctttaacattt gaaaatcaat caaaattcat attaacagaa taataatgaa 95880 aaaccatatgattttatata tatatatata ttttttttgt ttgtttgttt gttttgtttt 95940 ttttgttttttttttttgag acagtctcac tctccgccca ggctggagtg cattggtgct 96000 atctcagggctcaccgcaac ctctgcctgc tgggttcaat caattctgtc tcaacctcct 96060 gagtagctgggattataggt gcctgccacc atgcctagct aatttttgtg tttttagtag 96120 agatgaggtttcaccatgtt ggccaggatg gtctcaaact gctgacctca ggtgatccac 96180 ccgccttggcctcccaaagt gctaggatta caggtgtgag ccactgcacc tagccatgat 96240 tatcttaatagatgcacaca gcatttgaca aaatccaaca tccactcctg ctaaaaacac 96300 tgtacaaacaaggaatagaa ggaaacttcc tcaatccatt aaagggcacc tatgaaaatc 96360 ctacatttaatattatactt aatcacaatc aggaacaagg caagtatgtc cactgtcctt 96420 aattctattcaacattttac tgtaagttct acccagtgca ttaaggcaag aaaagaggta 96480 aaaggcatcaatattggaaa ggtagaagtg aaagtcttta tttaaaaaca tgagaatcta 96540 tgtagaaagtcctaaggagt ctaaaaaatg tgaatttagc aagtttgtaa ggtgtaaggg 96600 caatatatataaatcaattg tatttctgtg tggcaccagt gagcaattgg aaattgaaat 96660 gaaaaaccactaccatttac aatagcatca aacattgtga aaccttggga ataaacttgc 96720 aaaagacatgaaacctgcac actaaacact gcaaaatata gctgaaggaa attaaagaaa 96780 tcctgaataaatggagagag atgttaatgg atcataagat tcagtattgt tttcaatcta 96840 tagattcaaactgataaaaa tcccaggagg ctttttggta gaaattgata agctgattct 96900 taaaatcatgtgaaaatgca atggacatag aatagtcaaa acaactttga aaaagaacaa 96960 actgggaggacttacactac ctgatttaga agataatgtg gtattgatgt caacagaaac 97020 aaatagatcaatggaacaga gagtccagaa ataatctata caactacaga tgttcctcaa 97080 tttatgatggggtgatttcc caaaaaaccc atcttaagtt gaaaatattg ctagtcaaaa 97140 atatacttaacacacctaac ctactgaaca tcatagctta gcctagccta tctttttttt 97200 tttttttttttttttttgag acggagtctc gctctgtggc ccaggcggga gtgcagtggc 97260 gcaatctcggctcactgcaa gctccgcctc cagggttcac gccattctcc tgcctcagcc 97320 tccccagtagctgggactac aggcgcccac catcacgccc ggctaatttt ttttgtattt 97380 ttagtagagacggggtttca ccgtgttagc caggatggtc tcgatctcct gatctcgtga 97440 tccgcccgcctcggcctccc aaagtgctgg gattacaagc gtgagccacc gcgcccggcc 97500 agcctagcctatcttaaatg tgttcagaat acttacatta ccctgcagtt gggcaaaatc 97560 atctaatataaagcctattt tataatacag taatgaacat ttcatgtaat ttatggaata 97620 ctgaaagttactgtactgaa aaacgaaaaa acacaatggt tgtatgtgta ctggaagtac 97680 agtttctactgaatgcaaaa acttgcagct gagtgtgttc attatcttgg ttgtggtgat 97740 ggctccaccatgtatatgta tgtcaaagta catcaaatcg tacacgcaaa atatgtgcag 97800 gttattgcatgtcaggtata cctggatgaa tctgtaaaca atgtaatgaa agcaaaacaa 97860 aaagattaagagagcaaagt ttgtaggcta aatggaaaag aaataccacc aagcggggaa 97920 ccaaatcacagggtggaggc cctggaggat aagggtcagg agaggagaaa tgggggtagg 97980 tctcttaagtcaaaaggctg cgaacttctc tattccatgt taggatagca gagtttccaa 98040 gcgctgcatttggttgctgc tagatggcct tgccaggcta gataagcatt gggctgtctg 98100 acgatggtctcctgcatagt ttggtctcct gttttcctgt gtatgtgaca tgcttaagtt 98160 aggattatgtcactcaatca catctgcagt ggtacagcac gctagctggc caggtcgcgg 98220 tttgtcagtagtcatgtttt aaaagctgcc catttctggg ttatgcatat ctactaataa 98280 tggctataatatggaatgga aattaactgt gtcatccagc taaatttcag ctcagtttct 98340 ggtatgtatattaatgactt ctaaatacta aggatgtcaa aatgatttag atataatgct 98400 tttggtctagaatgggatat atactcaaat agttaatcaa aggtctgatc catggtgggc 98460 ttaagtggagaggcacatat ttctctcttg gggaggcaag ggaaaggacc acaacattct 98520 aactctctcagccaatcctc ttccactatg catatatagg ttgtgtggta cttggaattc 98580 ctgtatcatacttagccttt gatatggctc ttgagagtaa gagacaacag aaaaatgttg 98640 catttaacaacctgttacaa tgcttgttag agtgttttta taaactctaa ggtgttatgc 98700 aagtgtcatagttaataaaa tagcctaccc aacacccaac agacagactg gccatcttgc 98760 cacccaaatcctcccttgga tagaattaga gggggtatgg aatttaggaa ttagagtgta 98820 attaattacattgattatcc atagtcttta aaatatttta aattagaaac aagtctattt 98880 aaacagttttaagatttaca aaggatgaaa cttttcatta aatgaaagaa atagaggggt 98940 taagccaggaaatcctattt tacattaaga aaattattaa gagacactgg cttaaaccct 99000 agttccctctgagtttatag ggagagttcc catggagtgg gtgggtggag aagacaaaga 99060 catagatggatgctgatgag gaaagatgcg ggggtccttt tctgttgacc aagaacactg 99120 gggcaaagcacagttgaaca gcagcctgca gcctcacacc atggcacctt ttgagtccca 99180 tctgccctcatgtgctgggg gcaggaggtg gtgacagagg gcgtgggtca tggccagagg 99240 ttcctttcctcaaagcaaac aagcaaacgc cacatacggc tccccaaagc caggacttct 99300 tccctttggtcagtattctg ggacttctat tagcacatta gatttttctc atttatttgc 99360 cttcagtcaaggaaagctta tgttttcatc ctttgaacaa atcagacgtg gcaaatcttg 99420 aaggagaggtggctgtcccc caccactgtg ctgctcagaa tgtcaccagg tgggctggtg 99480 agaggagcacacagctgttc ccagctgata aaggggagag aagattgtgt ccttgatttt 99540 atttcactttctttggtatg tgtgaggcat ggtgccaaga tcttggtttt ttttgttttt 99600 tttttttaaactatacttct tccgtttcat caaaagtaat ttaattttgt tttacagtga 99660 atcctaactgatgtttttac ttttggggga tggagagggt gctatatttt tgtggttttc 99720 tgtgcctgactgggcagagc tttggatctt gtcccttgcc ccatgctgcc cagggcctgc 99780 cacttagcaagtactctgta gatatgtatt tgatgagcaa gggcctgagc atggatgtct 99840 gaggtgcaggcacgcactgc tgactggaga gccaggcagc agcatgggta ttcttcagca 99900 cagttcttttctgggagggt atttcttttc tatgtgatca atgagaacag gagtctccag 99960 gataattttatgtaagtcag tctttttgta tatacactgc ccccctaccc caccatatgt 100020 aaaatggatttcgcatatgc ctttccacaa ctgcagtgcc tcacctcccc aaaccgctgt 100080 ggctgatggactctgggccc caggtggagc tgtgctgccc ctacagcctg cagaaggccc 100140 agggtctggccttggcaatg actgtggttc gtgaagtggg taacacaatg acacatacgt 100200 gttctctgaggggaaacttc gttgcacaca gcccagggaa tttatgttat tgtaactttg 100260 gttctgaggcgttcttttat tattattatt actattattt ttagtaacag ctttattgtg 100320 atataattcatttaccatat aatttatcca tattaagtat acagttcaat gtttttagtt 100380 tattcacggtatgtggtgca accatcacca ccatcaattt tagaacattt tcatcacctg 100440 aaaagaaaccccatgcttct tagccatcat ttcccactcc ctatcccacc cacagcccta 100500 ggcaaccactaatttgcttt tctgactcta tggatttgcc tattctagac attttattat 100560 aaatggaatcatacaacatg tggtcctttg tgtctggctt attttgctta gcctgatgtt 100620 ttcaaggttcatctgtatca gtacctcatt ccttttcgta gctgaatact attccactgt 100680 atggatagaccacattttgt tgagccattc gtcagttagt ggacattcca cttttaggct 100740 gagttatgctgctatgaaca tttgtttata atctgaggat ttgtttttat attttcaatc 100800 tttgtcactttgaactgaga catgtacagg cacacaattt tggctccttt tggaattccc 100860 agacatagtattgcttgatg gcagcggaag tccatggagc acatgtcatg cagctgaaca 100920 cactacggggtagttaaaag gaagtacttg tttatgcaga tggggttaat tttagggaaa 100980 gtaagcttgaaataattttc tctgtacttt tgataatttt ctgtgtgtac ctaaaacata 101040 cattagcatgcatatttacc atttcaaata tgatgtgtgt ttggctaaaa aaaataaggg 101100 tctggccgggcacagtggct cacgcttgta atcccagcac tttgggaggc tgaggcaggc 101160 ggattgcgaggtcaggagtt tgagaccagc ctggccagca tggtgaaaca ctgtctctac 101220 tgaaaatacaaaaaatttgc tgggcatggt ggcgcatgcc tgtaatccca gctactaagg 101280 aggctgaggcaggagaattg cttgaaaccg ggaggcggag gttgcagtga gctgagattg 101340 taccactgcactccaggctg ggtgacagag tgagactctg tctcaagaaa agaaaaaaaa 101400 aaaaaggtctgtgccctcaa agcactcatg tccagtcttg ctgagggcag aagggtggct 101460 gtggggtgtgtgtggggaca aggcagacat ccagcatgtg gggcaacatg gtgtctctgc 101520 tgaggatgaacagggcactg tcagagtatc aggggacacc tcagaccaga cttagggtgg 101580 gatggtagggaggttggggg aagcttccag aaggaatttc tgaccaggtt ggaatctaca 101640 ggaggaatgggtataaatga gcaaaagaat cagggtagag aaagaggaag gagagagttt 101700 ccaagcagcaagttgagcat gttcggagca ccacacattc agggagttga gagggggact 101760 caaggcgaggtgtggtggga actgcagatg agagaagcgg ggagggccct ggtacctctg 101820 atagctgcaccaggtggttt ggactctatc ctatgagctg ggaagtcatt aaacggagcc 101880 ccatgagcagatctgctttt tggcctctca gaaggggaca cacggggcca agggtggggt 101940 cttgtttcccctgcggtggg agggcaagtc atctctgggg cgacagtggg aggtttgagg 102000 ctgtggggggattctggaag aaccaatgtg gagaacaaag tgagcacagg gattggagaa 102060 gcagcttcagggctattgaa aagatgaata ttttaaattc gtatcatcag acattatgga 102120 ggtccctagggatgtggcaa agcactacac ttacgtaatt gtgcttcaga atgtcccttg 102180 ccttacctgagttaaactta gttgaattga gctgccttaa ttgaactgaa agtgccaata 102240 aaaatagagaacaaaaactg ccaaaacaaa ttctgtggtt gctggagcac cagccatcat 102300 cagtctcatgacagccaaga ctcagcagct ccctggttga tttcacatat ttattcttgc 102360 tttgaaatggaaagcctgga agagaagcta attattaaag ggaatcaagg agtcaggcag 102420 gggtcggggggaggagattt atctgagctg ttactttgct gccattggga tgccacagta 102480 tctcaatcctagagttggag gggagttaaa cacagggcag ggcaggatgg gggaggcagc 102540 ctacccaggacgtggctgtg gggacctaag cagatgtgtt cctgcatgcg ttgctcagtg 102600 aggaactgaggctcagagag ctccagatgg tggctagaaa gtaggtctgt ctgactccaa 102660 atcagtggtcttcctgcccc agccaggtgc cactcaagcg agatgcagag gtggtagcag 102720 gggccctgccatggctggct gcggcacgtg gtacacacaa ggaggtggca gaggaggctt 102780 catcacattggccattcctt tgtttattaa actcccttta gatggggagc cctccgtggg 102840 gctaaaagtagaattaatct cactttctga ccatctctgt atctgttgct gcagatgaga 102900 aacaccacgtaatgatttcg ggagactaga tatactcgcc acggcaaggc cacaattatg 102960 ggcctggtggacacttcagg tggcaattta gtctgtctgc attaggccag gcttctcttc 103020 tagctctgtgacggggctgg ctctcaggga agatcccctg ggggaggtaa gaccatgctt 103080 ataagctcctgccacacatg cagctgtcaa agcaacccag atcacctcgg agcaggcgca 103140 cggaacagctgagcacacga cttctgctcc tttgctcaga gcaatgactt ctggctttta 103200 ttctttgtccaggtatgtac cctctggtca cagcgaccca ggatgctgac agcagccgga 103260 agctggctcacttgctgaac gccgtgaccg atgctttggt ttgggtgatt gccaagagcg 103320 gcatctcctcccagcagcaa tccatgcgcc tggctaacct cctgatgctc ctgtcccacg 103380 tcaggcatgcgaggtacgcg ccctaaggag ctgctctgct tgggcttggg atgggattat 103440 gtgctccacggagggtgaag tgatttggga aaagtgtctg caagttaagg aaaatgaatg 103500 cctgaaagggaatggggaat ttgtcagttc acacacctgt aagcaaagat gggcacagag 103560 tgggcatggaaggaatgtca tgtggtatct tacaggctct gcatggcagc cagtggtggc 103620 tcatgggtttttcaattgct ggggtttata gcctgtttat ggagtcctaa aaggggcagt 103680 tcctcccctaacacgaactg ccacccctgt ttacaccacc cagggctgag gccctgaggc 103740 cactttttgtggagaggcta agacccgctc ccctagatgg cccctcgagc tggtgatgcg 103800 aagaagtgcacaaatgcttc cctaagagtt gttctttcgg tggcatcagg aaattaagga 103860 taagacttaagagaagtggt ggacccagca gatttaggaa ggcagggctg taggtagggc 103920 atgtttctgatcaggaaacg taattgtgtg tgctgatgaa gagggtgtgc agtggtggct 103980 actgttggtacaatgatgct cagtgcttgg tgtcacccac gatgagggta gccttgccct 104040 ggagctggaggaggggaggg gagggtggaa ggtaattaac tggtcactga ggaggcaagt 104100 ctagaggctgtggagaagga caatatacac ctcgagaatc ttaagtgaga tgaagacctc 104160 tgcctttcccctttaatgat tgctcagcac atagccattt gcagaacaga tcctgtgttt 104220 gtagattccttcattgtgaa tttatctgct tgctaaaatt tatttgtaac cccaaaatca 104280 atatttgtggtgtttttgag gtcatgaaca gagtggcaga aattttgagt tgccctttat 104340 gtacagtcccagctgagatg gaacaagcag ctgctctcat actgtcaaca agtgtccttt 104400 acttggtctacttagtgcca tggttttaca tttttgtgct tttggtgact tcactgttta 104460 aaatgcccccctggtgtggt gctgaagacc tgtctagtgt tcctcggtgt gaaaaagctg 104520 tgatgtgccttatggagaaa gtatgtgtta agctttgctc gggtgtgagt tatagtgctg 104580 ctggccatgagttcaatgtt aatgagtcaa tggtatttat cacataaggc atctttagaa 104640 agaaacacacataaaacaag gttttgtatt gatcagctga tgaagatgtg gccagaggct 104700 tgcaggaacctaaccctgta tttcccctat gagtgaggat tcagtgttca cagtgacttt 104760 acggaacataattaccgcaa acaatgagga ttgattgtcc tatgtgtcag gccattgtag 104820 gtgtgtggtgggacacagag gctgacaaga catcgtcctt gcccttgagc ctaaattatc 104880 agggggagctggatgcacga gccatggata aatgggctgg gggaagagtg ggtttagggg 104940 tggggtagactggctctgag caaagagagc cggggaaggc ttcggggttc ctgtggctgc 105000 ctcggaggagggaatctcag cacctttttg tccccatagt aacaagggca tggaacatct 105060 gctcaacatgaagtgcaaaa atgtggtccc agtgtatgac ctgctgctgg agatgctgaa 105120 tgcccacgtgcttcgcgggt gcaagtcctc catcacgggg tccgagtgca gcccggcaga 105180 ggacagtaaaagcaaagagg gctcccagaa cccacagtct cagtgacgcc tggccctgag 105240 gtgaactggcccacagaggt cacaggctga agcgtgaact ccagtgtgtc aggagcctgg 105300 gcttcatctttctgctgtgt ggtccctcat ttggtgatgg caggcttggt catgtaccat 105360 ccttccctccaccttcccaa ctctcaggag tcggtgtgag gaagccatag tttcccttgt 105420 tagcagagggcacatttgaa tgcagcgttt ccacactcaa tggcctcata ggatctcagt 105480 gtggtctttcttactttcct tcttccttcc tcccctttgt gaaacatctt aaaggttttg 105540 gaatgaatggtggaaatctg acttggaagg gctgcgaatc agaaagggga gaggaagtga 105600 cacgcttacagaagtgggct aacccttctt gtgtggcaca cactaccctt ccctctgaga 105660 gttgacctttgctgttttcc ggaccactcc attgtaagat tgaaaacccc tgtggcaatt 105720 gcgtacttacctcccaggcc tgtggggact gatcatatca tatgatgctt attctgtcaa 105780 aggccagagggactgtggtt aagctgggat gtgagtcatg ttctctccct gaccttgctg 105840 ccagctgcacacagatttgt ccctctcgat ttgtattcac agagcctgcc aataatttgg 105900 ggtatgtgtgtatgagcgtg tgatcatttt catgcaggac tgtgggagat acaaatctcg 105960 ctgcttctggagctgctctt ccttaaacct gttgtcccat ggggccagcg tgggtgctgg 106020 agaaaggccgtgtttgcagg aatggggttc tctcctgtgg gtgtgggtga cagccacagt 106080 gtttccctggggcaatgtgg atgcagtttc catcttgtac aacctcataa gtagcagcca 106140 caattgccccatcagtcacc acaagtagtc agggatactt tgggctgtgg atgtgtgcag 106200 tgtgctgttttatggatgga tgagtagcta tgcaccccag tgtgtcagct ctggggccac 106260 actgtatagccttgatgagt acgccccttg aacaagaccc agtttgtgaa ctctccttaa 106320 agagaaatatttagggataa ttatttatag caagaaagaa ttcttttaca cttgagagct 106380 cttttaaaaatattttctta ttggaaaatt tatatggtgg gcagggtgaa aaagaaacag 106440 taaaaatattagttcttatt ccaagtggaa cataaatagg acatgaagaa gggcacctct 106500 gaaatgacaactttaactca ccttttaaaa gatgtgaaat ttccagtttt ggatacacgg 106560 tgaatatgtaaaatgagtaa cagcatacta tggaagccag caattaaata atcatgtttc 106620 attattgcagtaacgtttta aacaattacc ttgtgatatg atattaaata tattttcttt 106680 ttgaaaatatgttcactttg ggtagcacat cctgtattta ctaagtcatt aggaagactg 106740 cattcagtgttaccaagact ggtttttgct agtaagacct cgaataatcc ataattttga 106800 tattggtgcaattttactat aagttgagct tagctgtttc agaaatgctt ggacaagtac 106860 ctagagaacacactgatgtc tgtgttctga ggcagtctga agttattctt agagactcag 106920 ttacagctttagtaagattt agtacaggca ggataagctt ggtttcatag gaaccaggga 106980 accagtgttagtgtcagctt ctttcctcct ggtcagccta gaatccccca ctcccaatag 107040 aggggtttggaagctggaga gtaggaagta agaggcaaag aaggcagcct tcagcaactc 107100 attatctgccagtgaaattc tattaaatgt atttttaaaa gagattacca ggtaacaaaa 107160 acataaaaaaccaaaacaag gccagatgtg gtggctcacg cctgtaatcc cagcactttg 107220 ggaggccgaggtgggcgaac cacttgagcc catgagtttg actccaggct gggcaacatg 107280 gaaaccctgtcctacaaaag atacaaaaat tagccaggcg tggtggtgca ggcctgtagt 107340 tccagctacctgggaggctg aggtgggagg atcacctgag cctggggaga tcaaggctgc 107400 agtccattgcactccagcct gggtgacaga gggagaccct gtctcaaaaa aaaaaaaaaa 107460 aaaattgccacgaaatatat atatatatat atatataatt tttttttttt gagagtagat 107520 cttaagacagagatcacttc tactcctggg agtgaactgg caatggcaat ccctttagag 107580 cctcgagtgggcagtatcag gagcgccgca cagtgagttt ccagctgagc tattctcacc 107640 gaatctcgctctgttctcac agcacccctc tgtcaggcct gtctcatagt gactgcccac 107700 caggactgactacaaaagac ttgaccctaa aatagtcttg aagggatttt tctcaaaaaa 107760 ttaaggcgggaacacaagac aaagctgtca gcctagtcac aaatctgaag actcaactgc 107820 attaaaaatagtgcaaaatc ggcaggagct gtacagtgcg agtcttggtc tggaatactc 107880 cccctgctaactcagctgga agggcaacta tcttagattt cagtaaggaa gaaaaatcag 107940 ttaccaatacttggcagagc catattatat atccatatat atttatgtat ataagtggaa 108000 ttgaagcaattctagaattt tctagcatgt gaaagcaggg tttagttctt atttacgtct 108060 gctaagggacttttcaaatt caaagtgaac cttctgttta taggcctatt ttgaaacaaa 108120 gtatcctcacttaataagat ttgacacctt tttttttttt tttttgagac agggtctgac 108180 tcctgttgctcaggctagag tgcagtggcg ccatcatggc tcactgcagc ctcgacctcc 108240 tgggctcaagcgatcctccc accacagtgc cccatcccac cccattcccg ccctcgccga 108300 gtagctggggtgcacaccac cactcctggc taattctttt aatatttgta gagatggggt 108360 tttactatgctccccaggct ggtcctgaac tcctgggctc cagcgatctg cctgccaagg 108420 cctcccaaagtgctgagatt acaggcatga gccactgtgc ccagccccgc cacatttttt 108480 tttaagttgctgaaaatctt ttaaaaagat aaaaacacat tatttagtat ctaaagataa 108540 tatctgtgccagacacagtt ctcagtgcct cagacattca catttaatcc ttattataat 108600 aactgctatttccttatttt ctggttgtgg aactagacac ggtctaagca aacttgctga 108660 aggtcacgtggggagtaggt gattgagctg aacacaggca gtccaagtcc agtgctgaca 108720 gtgaccatgcacttcaaaca gtttaaaaat ttaaagaaaa atattttaaa actgcagaat 108780 ctatcaggtgcaacctgaca tgcacggctg ctgtgattta aatggggccc ccttgtgata 108840 cccccttacctcccaccaca atgtccagaa cacccctaca gacacagtaa gtttgtaaac 108900 ctctcacatcaaagttcaac tccacctttc atatctgtgt aaattaaagc ccacgggggc 108960 aaattcacctattcaaggtc ataaaactac tcatggcaaa gcttggactg gcacgcaagt 109020 cttctgcttgcctagcgggc cagtattgct cctgccccag gacttgcttc tgtgagaatc 109080 tgctttgtgagctgagtcgc agcagaatgg aggggcggtg aagttagggt tgtcttctgc 109140 tgtacctttagatcccatct cctcagctta gatgggtctg catgagcctt tacacaacag 109200 cagcaatgacagatggaaaa ataagatgca taatctgtta ttcccattgt cccatctcag 109260 gttcatgagctctagtgggt actgtgatca cctcctgtct gtgactgctt tccccaaaca 109320 cgtggaatatgttccttgga agtgtactca tgtaaaattc acatctttta ggcactgctg 109380 cttccctgtggagtgtgata tactacagtg tgaaaacacg tgccacttat tctttatagc 109440 tctcaaacttgctggaattt tggctccagt ggcagctctt aagatgtgca ttgtctgtga 109500 tgtatgatcgtagtgccatt tttgttgctt tggagtcagg gaggtttttt gtttgtttgt 109560 ttgtttgttttttaattccg aggatcctat tcacttgtag ggccagccac tggtaaactg 109620 gtggtgggtttcctctatgg gaagcacata aggagtggtg ataccagccg cgaacagttc 109680 ctgttaactgtacaatggat gtttttgcat ttgtttcctc tgttgggtgt ctaaatgcct 109740 taactgttggtcctatacct tttgtcattc aatgtgtact tcagagcctg ttggttggct 109800 ataatttgccattttctcag acgaatgctt tgtatcatta cactaatttg ttgacttcat 109860 ttgcaggctttacatttggg ccttgtagaa atgaatgttt gctgctctgt gaaagcagat 109920 tttgagacctgctttccctt cctccaggga gtgttttcct tactgtgtcc ctttaatgtc 109980 tatggcactgtcgtagagag tttaacatga tataaataaa gtgtttcatt attttggctt 110040 taaaaatgtatttgttgggg gttgagtgta agaacttaca gtaattaggc taagtagtgt 110100 ctacattctattctgaattc ttattgtggg gttagagagt cctttgagaa tttgatgaaa 110160 accagggctagtcttcctgg gaaagggcac ctgaacacaa atgcttgagt acaatttcag 110220 aagagttaagaagctctgct ttaatgtatc ttcttaaaaa gaacaatttc atctttagtc 110280 agctaatctcacacttgtga ttgatttatg accacaggtc ctgtgtatac aagtaaaatg 110340 cagctcacaaaagtcctggt atccagtgca tcgattattt ggatagattt tctgtaatca 110400 ttctgagtttgattagaatt atatccttta cagatgggga gaaaagcaat tcattcattt 110460 gaagttatcttagtgccaag agtcatgtga aaatgtccct tgcatgtggg caatgaaaga 110520 tttgcagacgatataaaacc cagactacct cataaaagag ttttgggaat acactgagct 110580 ttgagtgaaagaagctgcag tggcctccct ggagatgggg agcaaaccag cttaaaggcc 110640 cttatcctgaggaagagaca aaaattgaca tgcacaatat taagctttga aatgcagacc 110700 acacttcctttcactgcaac tttgacttgt cccgcatctc tacttaaggg cagaaaaggc 110760 ctctcaaacactcacctcat ttggaatgaa gatggagact cttttgcctg aagcaacgat 110820 ggagcagtgaccctctaatc aactcggtgg cctaaagaaa aatcttgggt aacattttca 110880 cttcagtttccctctgggat cattgtaatc catgaaaaaa ataattttaa agaaagagtt 110940 aaaatactttgaagttagtt atgtggttaa aaaccacctt cctttctatt atcaatccaa 111000 caatttgataactgtaaacg ctaaagtgaa gacggattct cttcagatgg tctccttaac 111060 tgcccagggcttgcagatgt ctcacccatg aggggcacca atgtagaaag ctgaggcttc 111120 atctactgatgagcttcact ggtttcccct gaggtttgtg ctttggcaga gaaggggagg 111180 aggggactgggattgtgtgg tcagctgtgc ctgccaacag atgcaggtta ggaactgtgt 111240 tcagtatcttccaataagaa aggggaaatg ccgatgccta tcctctttgt ttaggtagaa 111300 agtaaaatgctactggactt aaatgggcaa caaggggctt tgcctgttca tttgccatgg 111360 agagggctgggaatccaggt gcggtggctc acacctgtaa tcccaacact ttgggaggcc 111420 gaggtgggcagatcagttga ggtcaggagt ttgaaaccag cctggccaac atggcgaaac 111480 cccgtctctattaaaaatat aataattagc caggcatggt ggtgtgtgct tgtaatccca 111540 gctactcaggaggctgaggc atgagaatgg cttgaacctg gaaggcaaag gttgcagtga 111600 gccgagattgggccaccgca ctccagcctg ggtgactgac agagtgagac tctgtcaaaa 111660 aaaagagtagagtaaactgg gtataagatc cttccctttg cgtccacctc tcatgccatg 111720 ctgcctttgccattccctac aatagctgag ggtcacacgc tgaataattt aatttacaca 111780 tacacgagggtccagagcta agttaattct gtaaataaga cttagaataa aaggccctct 111840 ccaaatattttaaaaataat aatttttgtt ttttggaaga ttaagcatac cactgaactg 111900 ctttgttacagaattcagta caacagaagt ctggctaatt ttgtttttta atgagaaaca 111960 tctgagttgtacatatcaca aacagcttca agtttctgta ccaacccccc gcccccaccc 112020 ccgccgtggccaaacagtta aaacccaaag caaagcatca ctttggatgt gaaaaagtct 112080 tagaaaattaacttacaaaa acatccctat caagtcggta gtttggcatt tactttacat 112140 tagtcaaaagctccagctaa aatctaattt ttttaaaaaa aaatcgaagt ttacattatt 112200 catacagattgggcattgtt aaaaaatatg cacaaataac cacatccatg caatacaatt 112260 tctttaaaaatttaaagcaa tataaaagag cagagctagg tactgaacag aacattttgg 112320 tgtataaccggcagctcaaa attgccagct gattggagta aaactgattc taagcgtatt 112380 aaatatgattgattgtttcc atcagctaag ggtgcctatg agtttctgaa ccatttctag 112440 ggtggaatgtcctcgcttgc ttctataata tatgtgatgg acaccactgc tcattgacca 112500 tacctacattataataatgc tgttttacaa acaaaccaga attcacaaag tgcttggctc 112560 ttcaggaaactgacatttcc agagatccct aaactaatca actagttctg ccaaaatacc 112620 cggggcacctgccacacagg ttccctgctc ctggggagga acacaatctg aaagctgccc 112680 tgggctccagggagcccgtg ctgggtaagc ccagaagaag tctgcacagg tcccgggacc 112740 ttgccaacactaagtcactc agattggtct ggggccacgt gctgggcacc cttggcaatc 112800 aggcaggtggtgtagcactg tggccagcta tgccctctat gtggggggtg gcccattggt 112860 gtacctcagcatggggtaaa aggaccgggc aaagttgttg gcctgagtgc agctgtagtc 112920 ttcttcggaggagggcagca ggcaggccag gagcagcagc agcaggagga gcagctgcag 112980 gggtagggctgcccggacca 113000 17 644 DNA Homo sapiens exonexon junction(241)...(242) exon 3exon 4 17 tgttatgagg aaaaccccaa gagcatgctgccttacaaga caggtgaaaa atgtgttctg 60 tgaaagaaag agtaattaac tgttaaatgttacagactga tcaaataaaa tgaagactga 120 gaatggcctg tttgtaagat cacttttaaaaggaaaacat aggagcctga aacagaagtg 180 ggaaacaaat atttactcaa actaagagactaaactcagt agccagcaac aagagatcaa 240 gatggagtcc tcctctgtca cccaggctggaacgcagtgg tatgatctcg gctaactgca 300 acctcagcct gccaggttca agcaattcttctgcctcagc ctcccgagta gctgggatta 360 caggtgcctg ctgccatgat gattaattttatgtgttaac ttagctgggc tgtgttgccc 420 agataagttg gttaaacatt attctggatgtttctgtgaa gatgtttttg gatgaggtta 480 acatttagat cggtggactt tgagtaaagcagattacctt tcataatttg ggtggggctc 540 atcccaatca gttgaaccat ctgnaagagaccaaagactg accttctgca gcaagaaaaa 600 tctgccacag acaggccttg gactggacttccaccttgga tctc 644 18 575 DNA Homo sapiens exonexon junction(227)...(228) exonexon junction (322)...(332) exonexon junction(360)...(361) 18 aatatggttc tgaagacatc caagtggaga tatggcattt aaattcatgagattggatga 60 gatcccacca aaggaacagg tttaggtgga gacaaccaaa taccgatgcctaggacactg 120 cagtgtttag aattcaagga gatgagaagg aaacaggagg gaagattgaaaagaagagtc 180 cagtgtgtta tgaggaaaac cccaagagca tgctgcctta caagacagtggtccatcgcc 240 agttatcaca tctgtatgcg gaacctcaaa agagtccctg gtgtgaagcaagatcgctag 300 aacacacctt acctgtaaac agagagacac tgatgctcct gtcccacgtcaggcatgcga 360 gggcagaaaa ggcctctcaa acacccacct catttggaat gaagatggagactcttttgc 420 ctgaagcaac gatggagcag tgaccctctc atcaactcgg tggcctaaagaaaaatcttg 480 ggtaacattt tcacttcagt ttccctctgg gatcattgta atccatgaaaaaaataattt 540 taaagaaaga gttcaattaa aaaaaaaaaa aaaaa 575 19 20 DNAArtificial Sequence Antisense Oligonucleotide 19 tgtctccctc ttacaaacag20 20 20 DNA Artificial Sequence Antisense Oligonucleotide 20 cataggaggaaggtatgaaa 20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21gtgaccagag ggtacatact 20 22 20 DNA Artificial Sequence AntisenseOligonucleotide 22 ggtacatacc tgtccagaac 20 23 20 DNA ArtificialSequence Antisense Oligonucleotide 23 ggtacatacc gggaatcttc 20 24 20 DNAArtificial Sequence Antisense Oligonucleotide 24 ccctcatccc gggaatcttc20 25 20 DNA Artificial Sequence Antisense Oligonucleotide 25 ggactccatcttgatctctt 20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26atggaccact gtcttgtaag 20 27 20 DNA Artificial Sequence AntisenseOligonucleotide 27 agtgtctctc tgtttacagg 20 28 20 DNA ArtificialSequence Antisense Oligonucleotide 28 tttctgccct cgcatgcctg 20 29 20 DNAArtificial Sequence Antisense Oligonucleotide 29 gcttcccagg caatcgccca20 30 20 DNA Artificial Sequence Antisense Oligonucleotide 30 tgccgccgccctgtcaggct 20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31cttggatgtc ttcagaacca 20 32 20 DNA Artificial Sequence AntisenseOligonucleotide 32 cttctcatct ccttgaattc 20 33 20 DNA ArtificialSequence Antisense Oligonucleotide 33 agtaaatatt tgtttcccac 20 34 20 DNAArtificial Sequence Antisense Oligonucleotide 34 tgctggctac tgagtttagt20 35 20 DNA Artificial Sequence Antisense Oligonucleotide 35 aggactccatcttgatctct 20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36gcacctgtaa tcccagctac 20 37 20 DNA Artificial Sequence AntisenseOligonucleotide 37 cagaatttct aaagacttta 20 38 20 DNA ArtificialSequence Antisense Oligonucleotide 38 ctagagagtt ggttcagaat 20 39 20 DNAArtificial Sequence Antisense Oligonucleotide 39 ctgtaggcta caaactacct20 40 20 DNA Artificial Sequence Antisense Oligonucleotide 40 atcccatgttttctccactg 20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41cctgcacatc ccatgttttc 20 42 20 DNA Artificial Sequence AntisenseOligonucleotide 42 tctctcaaag tacccagtcc 20 43 20 DNA ArtificialSequence Antisense Oligonucleotide 43 tgaggcagag aagttagttt 20 44 20 DNAArtificial Sequence Antisense Oligonucleotide 44 cccaccctaa gtccaatttt20 45 20 DNA Artificial Sequence Antisense Oligonucleotide 45 tccaaagatggagaagcatc 20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46ttggtgttta gccaaaatag 20 47 20 DNA Artificial Sequence AntisenseOligonucleotide 47 tcagctgttt ggtgtttagc 20 48 20 DNA ArtificialSequence Antisense Oligonucleotide 48 agtaccagcc tcagctgttt 20 49 20 DNAArtificial Sequence Antisense Oligonucleotide 49 tctactcagg tggcataagg20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50 ccagcagcaaacgtaacctc 20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51gtgggtgtcc aaaaagccag 20 52 20 DNA Artificial Sequence AntisenseOligonucleotide 52 tccgcgcttg caactgcctc 20 53 20 DNA ArtificialSequence Antisense Oligonucleotide 53 cgcagctcgg gtggtccctc 20 54 20 DNAArtificial Sequence Antisense Oligonucleotide 54 gtataatggc ttgcagataa20 55 20 DNA Artificial Sequence Antisense Oligonucleotide 55 ttatatccatgtcttgagat 20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56agaatgtcat ggctggatat 20 57 20 DNA Artificial Sequence AntisenseOligonucleotide 57 ctgtttacag gtaaggtgtg 20 58 20 DNA ArtificialSequence Antisense Oligonucleotide 58 tctctctgtt tacaggtaag 20 59 20 DNAArtificial Sequence Antisense Oligonucleotide 59 cagtgtctct ctgtttacag20 60 20 DNA Artificial Sequence Antisense Oligonucleotide 60 cagaagtgagcatccctctt 20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61aaaggcctta catccttcac 20 62 20 DNA Artificial Sequence AntisenseOligonucleotide 62 ttgaatgctt cttttaaaaa 20 63 20 DNA ArtificialSequence Antisense Oligonucleotide 63 catggaggcc tcggtgaagg 20 64 20 DNAArtificial Sequence Antisense Oligonucleotide 64 atcatcatgg aggcctcggt20 65 20 DNA Artificial Sequence Antisense Oligonucleotide 65 gggacatcatcatggaggcc 20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66aggctgagct ccacaaagcc 20 67 20 DNA Artificial Sequence AntisenseOligonucleotide 67 gcaaagatga gcttgccggg 20 68 20 DNA ArtificialSequence Antisense Oligonucleotide 68 ctggagcaaa gatgagcttg 20 69 20 DNAArtificial Sequence Antisense Oligonucleotide 69 atgtcaaaga tttccagaat20 70 20 DNA Artificial Sequence Antisense Oligonucleotide 70 gggtacatactggaattgag 20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71gaccagaggg tacatactgg 20 72 20 DNA Artificial Sequence AntisenseOligonucleotide 72 tgagccagct tccggctgct 20 73 20 DNA ArtificialSequence Antisense Oligonucleotide 73 agcagatgtt ccatgccctt 20 74 20 DNAArtificial Sequence Antisense Oligonucleotide 74 ttctgggagc cctctttgct20 75 20 DNA Artificial Sequence Antisense Oligonucleotide 75 tcctcaggataagggccttt 20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76gcagtgaaag gaagtgtggt 20 77 20 DNA Artificial Sequence AntisenseOligonucleotide 77 actgctccat cgttgcttca 20 78 20 DNA ArtificialSequence Antisense Oligonucleotide 78 gcccctcatg ggtgagacat 20 79 20 DNAArtificial Sequence Antisense Oligonucleotide 79 tctgccaaag cacaaacctc20 80 20 DNA Artificial Sequence Antisense Oligonucleotide 80 gaagatactgaacacagttc 20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81tgttgcccat ttaagtccag 20 82 20 DNA Artificial Sequence AntisenseOligonucleotide 82 accgcacctg gattcccagc 20 83 20 DNA ArtificialSequence Antisense Oligonucleotide 83 ctggtttcaa actcctgacc 20 84 20 DNAArtificial Sequence Antisense Oligonucleotide 84 cggtggccca atctcggctc20 85 20 DNA Artificial Sequence Antisense Oligonucleotide 85 accacgcaccttcagaacca 20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86acttggatgt ctaagaggca 20 87 20 DNA Artificial Sequence AntisenseOligonucleotide 87 atgaagatgc ttaccagcca 20 88 20 DNA ArtificialSequence Antisense Oligonucleotide 88 tacatttgtt ttacaacact 20 89 20 DNAArtificial Sequence Antisense Oligonucleotide 89 gtataatggc tgtaaagaaa20 90 20 DNA Artificial Sequence Antisense Oligonucleotide 90 cagtgtctctctagggagca 20 91 20 DNA Artificial Sequence Antisense Oligonucleotide 91tttttctcac ctgtccagaa 20 92 20 DNA Artificial Sequence AntisenseOligonucleotide 92 ggacaattaa ttattggaaa 20 93 20 DNA ArtificialSequence Antisense Oligonucleotide 93 tgccctcata tcaaagattg 20 94 20 DNAArtificial Sequence Antisense Oligonucleotide 94 gcccaggctc ctgacacact20 95 20 DNA Artificial Sequence Antisense Oligonucleotide 95 aattgcttttctccccatct 20 96 20 DNA Artificial Sequence Antisense Oligonucleotide 96cttttctgcc cttaagtaga 20

What is claimed is:
 1. A compound 8 to 50 nucleobases in length targetedto a nucleic acid molecule encoding estrogen receptor beta, wherein saidcompound specifically hybridizes with said nucleic acid moleculeencoding estrogen receptor beta and inhibits the expression of estrogenreceptor beta.
 2. The compound of claim 1 which is an antisenseoligonucleotide.
 3. The compound of claim 2 wherein the antisenseoligonucleotide has a sequence comprising SEQ ID NO: 20, 21, 23, 24, 25,26, 27, 28, 29, 30, 36, 40, 43, 44, 48, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 88, 89, 90, 91, 92, 93, 94, 95 or96.
 4. The compound of claim 2 wherein the antisense oligonucleotidecomprises at least one modified internucleoside linkage.
 5. The compoundof claim 4 wherein the modified internucleoside linkage is aphosphorothioate linkage.
 6. The compound of claim 2 wherein theantisense oligonucleotide comprises at least one modified sugar moiety.7. The compound of claim 6 wherein the modified sugar moiety is a2′-O-methoxyethyl sugar moiety.
 8. The compound of claim 2 wherein theantisense oligonucleotide comprises at least one modified nucleobase. 9.The compound of claim 8 wherein the modified nucleobase is a5-methylcytosine.
 10. The compound of claim 2 wherein the antisenseoligonucleotide is a chimeric oligonucleotide.
 11. A compound 8 to 50nucleates in length which specifically hybridizes with at least an8-nucleobase portion of an active site on a nucleic acid moleculeencoding estrogen receptor beta.
 12. A composition comprising thecompound of claim 1 and a pharmaceutically acceptable carrier ordiluent.
 13. The composition of claim 12 further comprising a colloidaldispersion system.
 14. The composition of claim 12 wherein the compoundis an antisense oligonucleotide.
 15. A method of inhibiting theexpression of estrogen receptor beta in cells or tissues comprisingcontacting said cells or tissues with the compound of claim 1 so thatexpression of estrogen receptor beta is inhibited.
 16. A method oftreating an animal having a disease or condition associated withestrogen receptor beta comprising administering to said animal atherapeutically or prophylactically effective amount of the compound ofclaim 1 so that expression of estrogen receptor beta is inhibited. 17.The method of claim 16 wherein the disease or condition is cancer. 18.The method of claim 17 wherein the cancer is selected from the groupconsisting of leiomyoma, pancreatic cancer, prostate cancer, breastcancer, bone cancer and lymphoma.
 19. The compound of claim 1 targetedto a nucleic acid molecule encoding estrogen receptor beta, wherein saidcompound specifically hybridizes with and differentially inhibits theexpression of one of the variants of estrogen receptor beta relative tothe remaining variants of estrogen receptor beta.
 20. The compound ofclaim 19 targeted to a nucleic acid molecule encoding estrogen receptorbeta, wherein said compound hybridizes with and specifically inhibitsthe expression of a variant of estrogen receptor beta, wherein saidvariant is selected from the group consisting of ER-beta, ER-beta-2,ER-beta-3, ER-beta-4, ER-beta-5, ER-beta-6, ER-beta-7, ER-beta-8,ER-beta-9, ER-beta-5/6, ER-beta-cx and ER-beta-delta-2.